Sucrose transporter genes for increasing plant seed lipids

ABSTRACT

This invention relates to polynucleotide sequences encoding SUT2 or SUT4 sucrose transporter genes. Methods for increasing seed oil content and evaluating increased oil content in a plant seed are described. The compositions and methods disclosed herein employ a variety of sequences that encode sucrose transporters and a variety of sequences that influence fatty acid accumulation, including for example, DGAT, Lec1 and ODP1 transcription factor. In specific embodiments, overexpression of SUT2 and/or SUT4 sucrose transporters in combination with DGAT genes further increase plant seed oil production compared to high oil plant comprising recombinant DNA constructs that do not overexpress SUT2 or SUT4 transporters.

FIELD OF THE INVENTION

This invention is in the field of biotechnology; in particular, thispertains to polynucleotide sequences encoding SUT2 or SUT4 sucrosetransporters and the use of these disaccharide transporters forincreased seed lipid production in plants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of BB1784USDIV_CorrSeqLst.txt, a creation date of Apr. 13, 2015 anda size of 999 Kb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Plant lipids have a variety of industrial and nutritional uses and arecentral to plant membrane function and climatic adaptation. The primarystorage reserve of lipids in eukaryotic cells is in the form oftriacylglycerols (TAGs).

TAG is the primary component of vegetable oil in plants and is used bythe seed as a stored form of energy to be used during seed germination.The quality and content of plant oil can be altered by various methods,by impinging on the enzymes involved directly or indirectly in TAGbiosynthesis.

Most free fatty acids become esterified to coenzyme A (CoA) to yieldacyl-CoAs. These molecules are then substrates for glycerolipidsynthesis in the endoplasmic reticulum of the cell where phosphatidicacid and diacylglycerol (DAG) are produced. Either of these metabolicintermediates may be directed to membrane phospholipids (e.g.,phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) orDAG may be directed to form triacylglycerols (TAGS), the primary storagereserve of lipids in eukaryotic cells.

Sucrose is the major product of photosynthesis in higher plants and istransported from source to sink tissues through the phloem. It is alsothe major storage form of soluble carbon in sink tissues, and thereforealso serves as a long-term energy source. Sucrose transporters (SUTs)play a major role in the photoassimilate accumulation in the sinktissues. The SUTs have been categorized into three major subfamilies:Type I (SUT1), Type II (SUT2) and Type III (SUT4). (Kuhn, Plant Diol(2003) 5: 215-232; Lim et al., Physiologia Plantarum (2006) 16:572-584). Others have characterized sucrose transporters as highaffinity/low capacity, low affinity/high capacity, and mediumaffinity/high capacity transporters.

Altering the expression level of sucrose transporters can be expected tohave effects on the accumulation of photosynthetic assimilates in thesink tissues. Overexpression of a heterologous sucrose transporter hasbeen shown to increase sugar content in sink tissues such as potatotubers, but does not lead to change in starch content or tubermorphology (Leggewie et al. Planta (2003) 217: 158-167). Tissue specificoverexpression of heterologous sucrose transporters in storageparenchyma cells of pea cotyledons also increases sucrose influx intothese cells, and increases the growth rates of pea cotyledons but doesnot lead to an increase in dry weight of fully developed cotyledons(Rosche et al., Plant Journal (2002), 30(2): 165-175).

SUMMARY OF THE INVENTION

Methods and compositions are provided which modulate sucrose transportin a plant, plant cell or seed. Compositions are provided which compriseplants, plant cells and plant seeds having an increased oil content.Such compositions employ at least a first and a second polynucleotide.The first polynucleotide when expressed results in increased oil in saidplant and the second polynucleotide encodes a sucrose transporterpolypeptide. Various methods of use of such plants, plant cells andseeds are provided.

Further provided are methods which further increase oil content in ahigh oil plant seed, as well as, methods of evaluating oil content in aplant seed.

Additional compositions include polynucleotides and polypeptidesencoding sucrose transporters and active variants and fragments thereof.Plants, plant cells, and seeds comprising these sucrose transporters arefurther provided, as well as, various methods of use.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. § 1.822.

FIG. 1 shows sucrose levels recovered from yeast cells after varioustimes in culture for AtSUT2. Incubation time is shown on the x axis ashours (h) and μg sucrose/mg dry cell mass is shown on the v axis.

FIG. 2 shows sucrose levels recovered from yeast cells after varioustimes in culture for AtSUT4 and AtSUT4HIS. Incubation time is shown onthe x axis as hours (h) and μg sucrose/mg dry cell mass is shown on they axis.

FIG. 3 shows sucrose levels recovered from yeast cells after varioustimes in culture for AtSUC2 and AtSUC2HIS. Incubation time is shown onthe x axis as hours (h) and μg sucrose/mg dry cell mass is shown on they axis.

FIG. 4A-G presents an alignment of the amino acid sequences set forth inSEQ ID NOs: 4, 34, 36, 41-50, 52, and 150 of the SUT2 subfamily.

FIG. 5A-F presents an alignment of the amino acid sequences set forth inSEQ ID NOs: 53-69 of the SUT2 subfamily.

FIG. 6A-F presents an alignment of the amino acid sequences set forth inSEQ ID NOs: 6, 38, 40, 70-83, and 85 of the SUT4 subfamily.

FIG. 7 is a chart of the percent sequence identity and the divergencevalues for each pair of amino acids sequences presented in FIG. 4A-G.

FIG. 8 is a chart of the percent sequence identity and the divergencevalues for each pair of amino acids sequences presented in FIG. 5A-F.

FIG. 9 is a chart of the percent sequence identity and the divergencevalues for each pair of amino acids sequences presented in FIG. 6A-F.

FIG. 10 presents phylogenetic analysis of the SUT1, SUT2, and SUT4Arabidopsis thaliana genes and their homologs shown in Table 2 and Table3. Black circles represent members of the SUT4 family, black squaresrepresent members of the SUT1 family, and black triangles representmembers of the SUT2 family.

FIG. 11A-C presents an enlargement of the phylogenetic tree presented inFIG. 10. Dotted lines represent divisions between the SUT4 and SUT1families and also between the SUT1 and SUT2 families.

FIG. 12 presents oil increase by sucrose transporters co-expressed withYLDGAT1.

The sequence descriptions (Table 1) and Sequence Listing attached heretocomply with the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

TABLE 1 SEQ ID NO: Identifier Species Sequence Type 1 AtSUC2 (At1G22710)Arabidopsis thaliana DNA 2 AtSUC2(At1G22710) Arabidopsis thaliana PRT 3AtSUT2 (At2G02860) Arabidopsis thaliana DNA 4 AtSUT2(At2G02860)Arabidopsis thaliana PRT 5 AtSUT4 (At1G09960) Arabidopsis thaliana DNA 6AtSUT4(At1G09960) Arabidopsis thaliana PRT 7 YOL237 (AtSUC2 primer)Artificial sequence DNA 8 YOL132 (AtSUC2 primer) Artificial sequence DNA9 YOL174 (AtSUT2 primer) Artificial sequence DNA 10 YOL175 (AtSUT2primer) Artificial sequence DNA 11 YOL172 AtSUT4 primer Artificialsequence DNA 12 YOL173 AtSUT4 primer Artificial sequence DNA 13AtSUC2-pCRR2.1 Artificial sequence DNA 14 AtSUT2-pCR2.1 Artificialsequence DNA 15 AtSUT4-pCRR2.1 Artificial sequence DNA 16 AtSUC2HISArtificial sequence DNA 17 AtSUT2HIS Artificial sequence DNA 18AtSUT4HIS Artificial sequence DNA 19 YOL412 (AtSUC2 primer) Artificialsequence DNA 20 YOL413 (AtSUC2 primer) Artificial sequence DNA 21 YOL416(ATSUT2 primer) Artificial sequence DNA 22 YOL417 (AtSUT2 primer)Artificial sequence DNA 23 YOL414 (AtSUT4 primer) Artificial sequenceDNA 24 YOL415 (AtSUT4 primer) Artificial sequence DNA 25 RTW155(AtSUC2HIS6) Artificial sequence DNA 26 RTW156 (AtSUT4HIS6) Artificialsequence DNA 27 RTW157 (AtSUT2HIS6) Artificial sequence DNA 28 RTW247(yeast AtSUC2HIS) Artificial sequence DNA 29 RTW248 (yeast AtSUT4HIS)Artificial sequence DNA 30 RTW249 (yeast AtSUT2HIS) Artificial sequenceDNA 31 RTW250 (yeast AtSUC2) Artificial sequence DNA 32 RTW251 (yeastAtSUT4) Artificial sequence DNA 33 Glyma08g40980 (SUT2) Glycine max DNA34 Glyma08g40980 (SUT2) Glycine max PRT 35 Glyma18g15950 (SUT2) Glycinemax DNA 36 Glyma18g15950 (SUT2) Glycine max PRT 37 Glyma02g38300 (SUT4)Glycine max DNA 38 Glyma02g38300 (SUT4) Glycine max PRT 39 Glyma04g09460(SUT4) Glycine max DNA 40 Glyma04g09460 (SUT4) Glycine max PRT 41 CsSUT2(NCBI GI NO. 21063927) Citrus sinensis PRT 42 EuSUT2 (NCBI GI NO.61657989) Eucommia ulmoides PRT 43 StSUT2 (NCBI GI NO. 31096339) Solanumtuberosum PRT 44 LeSUT2 (NCBI GI NO. 10119908) Lycopersicon esculentumPRT 45 LOC_Os02g58080_SUC3 Oryza sativa PRT 46 cfp5n.pk008.k9_fis Zeamays PRT 47 Sb04g038030.1 Sorghum bicolor PRT 48 PmSUT2 (NCBI GI NO.31455370) Plantago major PRT 49 MeSUT2 (NCBI GI NO. 74476789) Manihotesculenta PRT 50 HbSUT2 (NCBI GI NO. 116008244) Hevea brasiliensis PRT51 Pn_Node_9230 Paspalum notatum DNA 52 Pn_Node_9230 Paspalum notatumPRT 53 ZmSUT1 (NCBI GI NO. 162463612) Zea mays PRT 54 Sb01g045720.1Sorghum bicolor PRT 55 cepe7.pk0015.d10 Zea mays PRT 56 TaSUT1A (NCBI GINO. 20152871) Triticum aestivum PRT 57 TaSUT1b (NCBI GI NO. 20152873)Triticum aestivum PRT 58 TaSUT1D (NCBI GI NO. 19548165) Triticumaestivum PRT 59 HvSUT1 (NCBI GI NO. 71890897) Hordeum vulgare PRT 60LOC_Os03g07480 Oryza sativa PRT 61 LOC_Os10g26470_SUC1 Oryza sativa PRT62 Sb01g022430.1 Sorghum bicolor PRT 63 cfp1n.pk007.b22_fis Zea mays PRT64 LOC_Os02g36700_BoSUT1 Oryza sativa PRT 65 Sb04g023860.1 Sorghumbicolor PRT 66 Sb07g028120.1 Sorghum bicolor PRT 67 BoSUT1_NCBI GI NO.66269698 Bambusa oldhamii PRT 68 cfp1n.pk065.p4_fis Zea mays PRT 69cfp3n.pk071.b8_fis Zea mays PRT 70 PsSUF4 (NCBI GI NO. 78192243) Pisumsativum PRT 71 HbSUT5 (NCBI GI NO. 118132673) Hevea brasiliensis PRT 72HbSUT4 (NCBI GI NO. 118132677) Hevea brasiliensis PRT 73 MeSUT4 (NCBI GINO. 74476785) Manihot esculenta PRT 74 VvSUC11 (NCBI GI NO. 6434829)Vitis vinifera PRT 75 StSUT4 (NCBI GI NO. 160425326) Solanum tuberosumPRT 76 DcSUT1a (NCBI GI NO. 2969887) Daucus carota PRT 77 ZmSUT4 (NCBIGI NO. 47571319) Zea mays PRT 78 Sb08g023310.1 Sorghum bicolor PRT 79LOC_Os12g44380_SUC4_or SUC2 Oryza sativa PRT 80 HvSUT2 (NCBI GI NO.7024413) Hordeum vulgare PRT 81 MdSUT4 (NCBI GI NO. 38327323) Malus ×domestica PRT 82 DgSUT4 (NCBI GI NO. 49609488) Datisca glomerata PRT 83LjSUT4 (NCBI GI NO. 28172870) Lotus japonicus PRT 84 Pn_Node_3980Paspalum notatum DNA 85 Pn_Node_3980 Paspalum notatum PRT 86 ATSUC8(At2G14670; NCBI GI NO. Arabidopsis thaliana PRT 15225986) 87 ATSUC7(At1G66570; NCBI GI NO. Arabidopsis thaliana PRT 115646796) 88 AtSUC6(At5G43610; NCBI GI NO. Arabidopsis thaliana PRT 15239921) 89 AtSUC9(At5G06170; NCBI GI NO. Arabidopsis thaliana PRT 15239949) 90 AtSUC1(At1G71880; NCBI GI NO. Arabidopsis thaliana PRT 56550707) 91 AtSUC5(At1G71890; NCBI GI NO. Arabidopsis thaliana PRT 15217602)custom5.pk301.c9 92 Glyma02g08250.1 Glycine max PRT 93 Glyma16g27320.1Glycine max PRT 94 PvSUT1 (NCBI GI NO. 78192247) Phaseolus vulgaris PRT95 sfl1.pk0001.g1_Glyma16g27340_&_27330 Glycine max PRT 96 PvSUT3 (NCBIGI NO. 78192251 Phaseolus vulgaris PRT 97 Glyma02g08260.1 Glycine maxPRT 98 sls2c.pk003.p4_Glyma16g27350.1 Glycine max PRT 99 PvSUF1 (NCBI GINO. 125625363) Phaseolus vulgaris PRT 100 SUF1_Ps (NCBI GI NO. 78192245)Pisum sativum PRT 101 Glyma10g36200.1 Glycine max PRT 102 PsSUT1 (NCBIGI NO. 5230818) Pisum sativum PRT 103 VfSut1 (NCBI GI NO. 1935019) Viciafaba PRT 104 HbSUT1 (NCBI GI NO. 116008246) Hevea brasiliensis PRT 105HbSUT6 (NCBI GI NO. 167859950) Hevea brasiliensis PRT 106 HbSUT3 (NCBIGI NO. 118132675) Hevea brasiliensis PRT 107 RcScr1 (NCBI GI NO. 468562)Ricinus communis PRT 108 PtSUT1 (NCBI GI NO. 77153413) Populus tremula ×PRT Populus tremuloides 109 EeSUT1 (NCBI GI NO. 7649151) Euphorbia esulaPRT 110 hss1c.pk009.b12_fis Helianthus annuus PRT 111 vs1n.pk016.e12_fisVernonia mespilifolia PRT 112 VvSUT27 (NCBI GI NO. 6434833) Vitisvinifera PRT 113 AmSUT1 (NCBI GI NO. 17447420) Alonsoa meridionalis PRT114 AbSUT1 (NCBI GI NO. 6120115) Asarina barclaiana PRT 115 NtSUT1a(NCBI GI NO. 575351) Nicotiana tabacum PRT 116 StSUT1 (NCBI GI NO.439294) Solanum tuberosum PRT 117 AgSUT2A (NCBI GI NO. 5566434) Apiumgraveolens PRT 118 AgSUT1 (NCBI GI NO. 4091891) Apium graveolens PRT 119DcSUT2 (NCBI GI NO. 2969884) Daucus carota PRT 120 BvSUT1 (NCBI GI NO.5823000) Beta vulgaris PRT 121 SoS21 (NCBI GI NO. 549000) Spinaciaoleracea PRT 122 CsSUT1 (NCBI GI NO. 21063921) Citrus sinensis PRT 123BoSUC2 (NCBI GI NO. 18091781) Brassica oleracea PRT 124 BoSUC1 (NCBI GINO. 18091779) Brassica oleracea PRT 125 PmSUC1 (NCBI GI NO. 667047)Plantago major PRT 126 NtSUT3 (NCBI GI NO. 4960089) Nicotiana tabacumPRT 127 YLDGAT1 Yarrowia lipolytica DNA 128 RTW218 Artificial sequenceDNA 129 KS349 Artificial sequence DNA 130 pKR268 Artificial sequence DNA131 RTW220 (AtSUC2HIS6 DGAT1 35Hyg) Artificial sequence DNA 132 RTW147(ANN-myb2 term sbf) Artificial sequence DNA 133 RTW158p1 (ann suc2)Artificial sequence DNA 134 RTW221 (ATSUT4HIS6 DGAT1 35Hyg) Artificialsequence DNA 135 RTW162p1 (ann sut4) Artificial sequence DNA 136 RTW222(ATSUT2HIS6 DGAT1 35Hyg) Artificial sequence DNA 137 RTW166p1 (ann SUT2)Artificial sequence DNA 138 RTW212(plasmid with soybean Artificialsequence DNA selection marker) 139 RTW226 Artificial sequence DNA 140RTW227F (ATSUT4 DGAT1 ALS), Artificial sequence DNA soy expression clone141 GmSut2-1For (Glyma08g40980 Artificial sequence DNA primer) 142GmSut2-1Rev (Glyma08g40980 Artificial sequence DNA primer) 143GmSut2-2For (Glyma18g15950 Artificial sequence DNA primer) 144GmSut2-2Rev (Glyma18g15950 Artificial sequence DNA primer) 145 SA150(Glyma02g38300 primer) Artificial sequence DNA 146 SA151(Glyma02g38300primer) Artificial sequence DNA 147 SA148 (Glyma04g09460 primer)Artificial sequence DNA 148 SA149 (Glyma04g09460 primer) Artificialsequence DNA 149 Pn_Node_40538 Paspalum notatum DNA 150 Pn_Node_40538Paspalum notatum PRT 151 Glyma02g38300 in pGEM-T Easy Artificialsequence DNA 152 pKR1680 Artificial sequence DNA 153 pKR1684 Artificialsequence DNA 154 pLF235 Artificial sequence DNA 155 pKR1681 Artificialsequence DNA 156 pKR1685 Artificial sequence DNA 157 GmSut4-2ForArtificial sequence DNA 158 GmSut4-2Rev Artificial sequence DNA 159pLF236 Artificial sequence DNA 160 pKR1682 Artificial sequence DNA 161pKR1686 Artificial sequence DNA 162 pKR1468 Artificial sequence DNA 163pKR1691 Artificial sequence DNA 164 pKR1698 Artificial sequence DNA 165pKR1699 Artificial sequence DNA 166 pKR1700 Artificial sequence DNA 167pKR1701 Artificial sequence DNA 168 pKR1363 Artificial sequence DNA 169pKR1331 Artificial sequence DNA 170 pKR1365 Artificial sequence DNA 171pKR1374 Artificial sequence DNA 172 pKR1598 Artificial sequence DNA 173pKR1600 Artificial sequence DNA 174 pKR1602 Artificial sequence DNA 175pKR1658 Artificial sequence DNA 176 pKR1661 Artificial sequence DNA 177hso1c.pk009.l6:fis Helianthus annuus DNA 178 hso1c.pk009.l6:fis:+2 Frame+2 Helianthus annuus PRT translation 179 YLDGAT2 Yarrowia lipolytica DNA180 YLDGAT2 Yarrowia lipolytica PRT 181 YLDGAT1 gene codon optimized forArtifical Sequence DNA soybean 182 YLDGAT1 gene codon optimized forArtifical Sequence PRT soybean 183 YLDGAT2 gene codon optimized forArtifical Sequence DNA soybean 184 YLDGAT2 gene codon optimized forArtifical Sequence PRT soybean 185 YLDGAT2 comprising codon 326Artifical Sequence DNA mutated from Tyr to Phe 186 YLDGAT2 comprisingcodon 326 Artifical Sequence PRT mutated from Tyr to Phe 187 YLDGAT2comprising codon 326 Artifical Sequence DNA mutated from Tyr to Leu 188YLDGAT2 comprising codon 326 Artifical Sequence PRT mutated from Tyr toLeu 189 YLDGAT2 comprising codon 327 Artifical Sequence DNA mutated fromArg to Lys 190 YLDGAT2 comprising codon 327 Artifical Sequence PRTmutated from Arg to Lys 191 YLDGAT1 Yarrowia lipolytica PRT

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current invention includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current invention includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

As used herein, the term plant also includes plant protoplasts, plantcell tissue cultures from which plants can be regenerated, plant clumps,and plant cells that are intact in plants or parts of plants such asembryos, pollen, ovules, flowers, branches, fruit, kernels, ears, cobs,husks, stalks, roots, root tips, anthers, and the like. Grain isintended to mean the mature seed produced by commercial growers forpurposes other than growing or reproducing the species. Progeny,variants, and mutants of the regenerated plants are also included withinthe scope of the invention, provided that these parts comprise theintroduced polynucleotides.

In one embodiment, any plant species may be utilized in the invention,including, but not limited to, monocots and dicots. Examples of plantsthat may be used in the invention include, but are not limited to, corn(Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago saliva), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Thiticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumharbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables of interest include, but are not limited to, tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum.

Conifers of interest that may be employed in practicing the presentinvention include, but are not limited to, pines such as loblolly pine(Pinta taeda), slash pine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Hardwoodtrees can also be employed including ash, aspen, beech, basswood, birch,black cherry, black walnut, buckeye, American chestnut, cottonwood,dogwood, elm, hackberry, hickory, holly, locust, magnolia, maple, oak,poplar, red alder, redbud, royal paulownia, sassafras, sweetgum,sycamore, tupelo, willow, yellow-poplar.

In specific embodiments, plants of the present invention are crop plants(for example, corn, alfalfa, sunflower, Brassica, soybean, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In otherembodiments, corn and soybean and sugarcane plants are optimal, and inyet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Other plants of interest including Turfgrasses such as, for example,turfgrasses from the genus Poa, Agrostis, Festuca, Lolium, and Zoysia.Additional turfgrasses can come from the subfamily Panicoideae.Turfgrasses can further include, but are not limited to, Blue gramma(Bouteloua gracilis (H.B.K.) Lag. Ex Griffiths); Buffalograss (Buchloedactyloids (Nutt.) Engelm.); Slender creeping red fescue (Festuca rubrassp. Litoralis); Red fescue (Festuca rubra); Colonial bentgrass(Agrostis tenuis Sibth.); Creeping bentgrass (Agrostis palustris Huds.);Fairway wheatgrass (Agropyron cristatum (L.) Gaertn.); Hard fescue(Festuca longifolia Thuill.); Kentucky bluegrass (Poa pratensis L.);Perennial ryegrass (Lolium perenne L.); Rough bluegrass (Poa trivialisL.); Sideoats grama (Bouteloua curtipendula Michx. Torr.); Smoothbromegrass (Bromus inermis Leyss.); Tall fescue (Festuca arundinaceaSchreb.); Annual bluegrass (Poa annua L.); Annual ryegrass (Loliummultiflorum Lam.); Redtop (Agrostis alba L.); Japanese lawn grass(Zoysia japonica); bermudagrass (Cynodon dactylon; Cynodon spp. L.C.Rich; Cynodon transvaalensis); Seashore paspalum (Paspalum vaginatumSwartz); Zoysiagrass (Zoysia spp. Willd; Zoysia japonica and Z. matrellavar. matrella); Bahiagrass (Paspalum notatum Flugge); Carpetgrass(Axonopus affinis Chase); Centipedegrass (Eremochloa ophiuroides MunroHack.); Kikuyugrass (Pennisetum clandesinum Hochst Ex Chiov); Browntopbent (Agrostis tenuis also known as A. capillaris); Velvet bent(Agrostis canina); Perennial ryegrass (Lolium perenne); and, St.Augustinegrass (Stenotaphrum secundatum Walt. Kuntze). Additionalgrasses of interest include switchgrass (Panicum virgatum).

“Progeny” comprises any subsequent generation of a plant.

“Regeneration” or “regenerated” plants is intended to mean thattransformed plant cells may be grown into plants in accordance withconventional ways. See, for example, McCormick et al. (1986) Plant CellReports 5:81-84. These plants may then be grown, and either pollinatedwith the same transformed strain or different strains, and the resultingprogeny having constitutive expression of the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native foini in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably to refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Coding region” refers to a polynucleotide sequence that whentranscribed, processed, and/or translated results in the production of apolypeptide sequence.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment Isolated polynucleotides may be purifiedfrom a host cell in which they naturally occur. Conventional nucleicacid purification methods known to skilled artisans may be used toobtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” or “regulatory elements” are used interchangeablyand refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence, Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences,The terms “regulatory sequence” and “regulatory element” are usedinterchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably to refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in a nullsegregating (or non-transgenic) organism from the same experiment.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid sequence orfragment (e.g., a recombinant DNA construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid sequence orfragment may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid sequence orfragment (e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, a desirable phenotype, e.g., increased cell walldigestibility, or alternatively, is an allele that allows theidentification of plants with decreased cell wall digestibility that canbe removed from a breeding program or planting (“counterselection”). Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype, or alternatively, segregates with the unfavorableplant phenotype, therefore providing the benefit of identifying plants.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid sequence orfragment (e.g., a recombinant DNA construct/expression construct) into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid fragment intoa eukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, include lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an anti sense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onoverexpression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the overexpressed sequence (see Vaucheret et al.,Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication No.WO 98/36083 published on Aug. 20, 1998).

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PIGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., Trends Genet.15:358 (1999)).

Small RNAs play an important role in controlling gene expression.Regulation of many developmental processes, including flowering, iscontrolled by small RNAs. It is now possible to engineer changes in geneexpression of plant genes by using transgenic constructs which producesmall RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA orDNA target sequences. When bound to RNA, small RNAs trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, it is thought that small RNAs can mediate DNAmethylation of the target sequence. The consequence of these events,regardless of the specific mechanism, is that gene expression isinhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides (nt) in length that have been identified in both animals andplants (Lagos-Quintana et al., Science 294:853-858 (2001),Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al.,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001);Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes.Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002);Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processedfrom longer precursor transcripts that range in size from approximately70 to 200 nt, and these precursor transcripts have the ability to formstable hairpin structures.

MicroRNAs (miRNAs) appear to regulate target genes by binding tocomplementary sequences located in the transcripts produced by thesegenes. It seems likely that miRNAs can enter at least two pathways oftarget gene regulation: (1) translational inhibition; and (2) RNAcleavage. MicroRNAs entering the RNA cleavage pathway are analogous tothe 21-25 nt short interfering RNAs (siRNAs) generated during RNAinterference (RNAi) in animals and posttranscriptional gene silencing(PTGS) in plants, and likely are incorporated into an RNA-inducedsilencing complex (RISC) that is similar or identical to that seen forRNAi.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Multiple alignment of the sequences provided herein wereperformed using the Clustal W method of alignment (Thompson, J. D., etal. (1994) Nucleic Acids Research 22: 4673-80) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DEVERGENT SEQS(%)=30%, DNA TRANSITION WEIGHT=0.5, PROTEIN WEIGHT MATRIX “GonnetSeries”). Default parameters for pairwise alignments using the Clustal Wmethod were SLOW-ACCURATE, GAP PENALTY=10, GAP LENGTH=0.10, PROTEINWEIGHT MATRIX “Cionnet 250”.

After alignment of the sequences, using the Clustal W program, it ispossible to obtain “percent identity” and “divergence” values by viewingthe “sequence distances” table on the same program; unless statedotherwise, percent identities and divergences provided in the figureswere calculated in this manner. In specific embodiments, the varioussequences employed in the various methods and compositions disclosedherein can be aligned to the recited SEQ ID NOs based on the Clustal Wmethod of alignment with pairwise alignment default parameters. Foramino acid sequences the following parameters are used: KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acid sequencesthe following parameters are used: KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4.

It is recognized that sequence alignments and percent identitycalculations may be determined using other mathematical algorithms whichwould be known to those of ordinary skill in the art. Non-limitingexamples of such mathematical algorithms are the algorithm of Myers andMiller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith etal. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: the ALIGN program(Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCGWisconsin Genetics Software Package, Version 10 (available from AccelrysInc., 9685 Scranton Road, San Diego, Calif., USA). Alignments usingthese programs can be performed using the default parameters. The ALIGNprogram is based on the algorithm of Myers and Miller (1988) supra. APAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intel ligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

The compositions and methods disclosed herein employ a variety ofsequences that encode sucrose transporters and a variety of sequencesthat influence fatty acid accumulation, including for example, DGAT,Lec1 and ODP1 transcription factor. Variant polynucleotides andpolypeptides of these sequences are provided. Variants of suchpolynucleotides (i.e, sequences that encode the sucrose transporters orthe sequences that influence fatty acid accumulation (i.e., the SUT2,SUT4, DGAT, lec1 and ODP1 sequences), and polypeptides encoded thereby,can be employed in the methods and compositions disclosed herein. Asused herein, “variants” is intended to mean substantially similarsequences. For polynucleotides, a variant comprises a polynucleotidehaving deletions (i.e., truncations) at the 5′ and/or 3′ end; deletionand/or addition of one or more nucleotides at one or more internal sitesin the native polynucleotide; and/or substitution of one or morenucleotides at one or more sites in the native polynucleotide. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the sucrose transporters or the sequences thatinfluence fatty acid accumulation (i.e., the SUT2, SUT4, DGAT, lec1 andODP1 sequences). Naturally occurring allelic variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant polynucleotides also includesynthetically derived polynucleotides, such as those generated, forexample, by using site-directed mutagenesis but which still encode oneof the sucrose transporters or the sequences that influence fatty acidaccumulation (i.e., the SUT2, SUT4, DGAT, lec1 and ODP1 sequences).Generally, variants of a particular polynucleotide of the invention aSUT2, SUT4 and/or a DGAT, Led or ODP-1) will have at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide (i.e., to any one of SEQ ID NOS: 1, 3, 5, 33, 35, 37, 39,127, 177, 179, 181, 183, 185, 187, or 189 as determined by sequencealignment programs and parameters as described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 4, 6, 34, 36, 38, 40-50, 52-83, 178, 180, 182,184, 186, 188, 190, or 191 is disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

As used herein, a “variant” polypeptide is intended to mean apolypeptide derived from the native polypeptide or by deletion(so-called truncation) of one or more amino acids at the N-terminaland/or C-terminal end of the native polypeptide; deletion and/oraddition of one or more amino acids at one or more internal sites in thenative polypeptide; or substitution of one or more amino acids at one ormore sites in the native polypeptide. Variant polypeptides encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native polypeptide,that is, sucrose transport activity as described herein, influence oilaccumulation, or have DGAT, ODP-1 or Led activity. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of the sucrose transporters(i.e., SUT2 or SUT4) or of the sequences that influence fatty acidaccumulation (i.e., DGAT, lec1 and ODP1 sequences) employed in thevarious methods and compositions disclosed herein will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the polypeptide (i.e., to any one of the amino acidsequences set forth in 4, 6, 34, 38, 40-50, 52-83, 178, 180, 182, 184,186, 188, 190 or 191 as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa polypeptide of the invention may differ from that polypeptide by asfew as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as5, as few as 4, 3, 2, or even 1 amino acid residue.

The polypeptides employed herein (i.e., the sucrose transporters or thesequences that influence fatty acid accumulation (i.e., the SUT2, SUT4,DGAT, lec1 and ODP1 sequences)) may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the sucrosetransport polypeptides of the invention can be prepared by mutations inthe DNA. Methods for mutagenesis and polynucleotide alterations are wellknown in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Thus, the polynucleotides employed herein include both the naturallyoccurring sequences as well as mutant forms. Likewise, the polypeptidesof the invention encompass naturally occurring polypeptides as well asvariations and modified forms thereof. Such variants will continue topossess sucrose transport activity as described herein, influence oilaccumulation, or have DGAT, ODP-1 or Lec1 activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the sucrose transportersor the sequences that influence fatty acid accumulation (i.e., the SUT2,SUT4, DGAT, lec1 and ODP1 sequences) encompassed herein are not expectedto produce radical changes in the characteristics of the polypeptide.However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays. That is, the activity can be evaluated by functionalassays of sucrose transporter genes. See, for example, Example 2 of theinvention as described herein below. Methods of evaluating oil contentare described in Examples 7, 8, 14, 15 and 16.

Variant polynucleotides and polypeptides also encompass sequences andpolypeptides derived from mutagenic and recombinogenic procedures suchas DNA shuffling. With such a procedure, one or more different sucrosetransporter coding sequences can be manipulated to create new sucrosetransporter polypeptides possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Nall. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

A “high oil plant” is defined as a transgenic plant having higher oilcontent when compared to a non-transgenic or null segregant plant. Thetransgenic plant comprises in its genome at least one recombinant DNAconstruct comprised of a polynucleotide operably linked to at least oneregulatory element. The polynucleotide can encode a protein that isinvolved in fatty acid accumulation. Examples of such a protein include,but are not limited to: DGAT, Lec1 and ODP1 transcription factor.Alternatively, expression of the polynucleotide can result in silencingof an expressed gene resulting in fatty acid accumulation. Examples ofsaid gene include, but are not limited to, phosphoglucomutase (PGM).

A high oil plant can be obtained by various ways, examples of whichinclude, but are not limited to: selection of naturally occurringallelic variant plants that consistently exhibit oil content higher thanthe control, mutagenesis followed by selection based on increase in oilcontent above the control plants, and any manipulation that induces orrepresses activity of a gene that results in a high oil plant.

The plant with naturally occurring allelic variants for high oilphenotype can be, but is not limited to, Arabidopsis (Hobbs et al, PlantPhysiology (2004) 136(2): 3341-3349), corn (US application Ser. No.11/680,922; Zheng et al, (2008) Nature Genetics, 40(3); 367-372), canola(Delourme et al., (2006) Theoretical and Applied Genetics 113(7):1331-1345), and soybean (Panthee et al., (2005) Crop Science 45(5):2015-2022).

In the context of this invention, a high oil line is any plant that whena sucrose transporter is overexpressed, a further increase of oilresults as compared to a plant that does not have overexpression of asucrose transporter gene.

A high oil plant can include any plant with an increase in the level ofoil in the plant or plant part, for example, in the seed or kerneland/or the embryo or germ, or any combination thereof. For example,increased oil content can comprise an increase in overall oil level inthe plant or plant part of about 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 120%, or greater when compared to acontrol plant or plant part. Alternatively, the increased level of oilcan include about a 0.5-fold, 1-fold, 2-fold, 4-fold, 8-fold, 16-fold,32-fold, or greater overall increase in oil level in the plant or theplant part when compared to a control plant or plant part. See, U.S.application Ser. No. 11/680,922, herein incorporated by reference in itsentirety.

“Diacylglycerol acyltransferase” or “DGAT” is an integral membraneprotein that catalyzes the final enzymatic step in the production oftriacylglycerols in plants, fungi and mammals. This enzyme isresponsible for transferring an acyl group from acyl-coenzyme-A to thesn-3 position of 1,2-diacylglycerol (“DAG”) to forni triacylglycerol(“TAG”). DGAT is associated with membrane and lipid body fractions inplants and fungi, particularly, in oilseeds where it contributes to thestorage of carbon used as energy reserves. DGAT is known to regulate TAGstructure and direct TAG synthesis. Furthermore, it is known that theDGAT reaction is specific for oil synthesis. Active variants andfragments of a DGAT polypeptide when expressed in a plant or plant seedwill therefore increase the oil content of a plant seed.

Two different families of DGAT proteins have been identified. The firstfamily of DGAT proteins (“DGAT1”) is related to the acyl-coenzymeA:cholesterol acyltransferase (“ACAT”) and has been described in U.S.Pat. Nos. 6,100,077 and 6,344,548, both of which are herein incorporatedby reference. A second family of DGAT proteins (“DGAT2”) is unrelated tothe DGAT1 family and is described in PCT Patent Publication WO2004/011671 published Feb. 5, 2004 and herein incorporated by reference.Other references to DGAT genes and their use in plants include PCTPublication No. W01998/055,631 and U.S. Pat. No. 6,822,141, each ofwhich is herein incorporated by reference.

“DGAT” and diacylglycerol acyltransferase are used interchangeablyherein and refer to any member, or combination, of the DGAT1 or DGAT2family of proteins.

Plant and fungal DGAT genes have been described previously (U.S. Pat.Nos. 7,198,937 and 7,465,565, US Publication No. 2008/0295204, U.S.application Ser. Nos. 12/470,569 and 12/470,517). Each of thesereferences is herein incorporated by reference. Non-limiting examples ofDGAT sequences and active variant thereof from Yarrowia lipolytica areset forth in SEQ ID NO: 127 and 179-191.

Leafy cotyledon) or Lec1/Hap3 is a key regulator of seed development inplants. Lec1 is a CCAAT-binding factor (CBF)-type transcription factor.The terms leafy cotyledon 1, Hap 3, Lec1, and Hap3/Lec1 are usedinterchangeably herein and refer to a class of transcription factors.U.S. Pat. No. 6,235,975 describes leafy cotyledon1 genes and their uses.A pending U.S. patent application (U.S. application Ser. No. 11/899,370)relates to isolated nucleic acid fragments encoding Lec1 relatedtranscription factors. Issued patent (U.S. Pat. No. 7,294,759) describesthe use of Lec1 genes for altering oil content in plants. Each of theseapplications is herein incorporated by reference in their entirety. InArabidopsis, Lec1 has been shown to regulate the expression of fattyacid biosynthetic genes (Mu et al., Plant Physiology (2008) 148:1042-1054).

Both starch and fatty acid biosynthesis occur in plastids. Thesebiosynthetic pathways compete for the same precursors such asglucose-6-phosphate. Phosphoglucomutase (PGM) that facilitates theinterconversion of glucose-6-phosphate (G6P) and glucose-1-phosphate isan important regulator of these pathways (Periappuram et al., (2000)Plant Physiol. 122:1193-1199). Also there are plastidic and cytosolicforms of phosphoglucomutase and both catalyze the conversion ofglucose-6-phosphate to glucose 1-phosphate in different subcellularlocations.

Previous reports on a plastidic PGM mutant (pgm-1) from the oilseedplant Arabidopsis (Caspar et al., (1985) Plant Physiol. 79:11-17;Periappuram et al., (2000) Plant Physiol. 122:1193-1199) indicated thatpgm-1 mutant plants showed a decrease in seed lipid content and anincrease in leaf soluble carbohydrates. High levels of solublecarbohydrates were also observed in starchless Nicotiana sylvestrisplants deficient in the plastidic PGM activity (Huber and Hanson, (1992)Plant Physiol. 99:1449-1454). Yet another effect of reduced starchcontent on carbon partitioning was observed in pea (Pisum sativum).Seeds from wild type pea typically contain 60% of the seed dry weight asstarch. The rug3 locus of Pisum sativum encodes the pea plastidicphosphoglucomutase. Pea seeds, of the rug3rug3 genotype, substantiallylacking plastidic phosphoglucomutase activity, have a wrinkledphenotype, higher levels of sucrose and an increased lipid content atmaturity (EP No. 1001029A1; Casey et al., (1998) J. Plant Physiol. 152:636-640),

Issued U.S. Pat. Nos. 7,250,557 and 7,323,560 respectively describeisolated plastidic phosphoglucomutase nucleic acids and methods of useof these nucleic acids in altering oil content in plants. Also, U.S.application Ser. No. 12/470,509 describes transgenic plants with alteredDGAT and PGM expression profiles to achieve increased oil content anddifferent fatty acid expression profiles. Each of these references isherein incorporated by reference in their entirety.

Ovule Development Protein (ODP) is a transcription factor containing twoAP2 domains. AP2 transcription factors (herein referred tointerchangeably as “AP2 domain transcription factors”, “AP2 proteins”,or “AP2 transcription factor proteins”) such as ODP, activate severalgenes in the oil or TAG biosynthetic pathway in the plant cell. USPRV61/165,548 describes the use of an ODP-1 gene for alteration of oiltraits in plants. U.S. Pat. No. 7,579,529 describes an AP2 domaintranscription factor and methods of its use. U.S. Pat. No. 7,157,621discloses the use of ODP transcription factor for increasing oil contentin plants. Each of these references is herein incorporated by referencein their entirety.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

“Sucrose transporters” (SUTs) are polynucleotides that encode a class ofsucrose/H+ symporters that facilitate transport of sucrose across plantmembranes in various plant tissues. Sucrose transporters are present inmany plants including, but not limited to, maize, spinach, potato,tomato, pea, Arabidopsis, celery, grape, tobacco, Lotus, broad bean, andrice (for a review see Kuhn, C. (2003) Plant biol. 5:215-232; Allen etal. U.S. Pat. No. 7,288,645). The terms “sucrose transporter” and SUTare used interchangeably herein. Three subfamilies of SUTs are known inplants. The SUT1 subfamily is defined as high affinity, low capacitytransporters; the SUT2 subfamily is defined as low affinity or very lowaffinity, high capacity transporters; and, the SUT4 subfamily is definedas medium or low affinity, high capacity transporters (Rosche et al.(2002) The Plant Journal 30(2): 165-175; Kuhn, C. (2003) Plant biol.5:215-232; Sauer, N. (2007) FEBS Letters 581:2309-2317; Lalonde et al.(2004) Ann. Rev. Plant Biol. 55:341-372). The SUT1 subfamily of highaffinity transporters is not relevant to this invention. AtSUT2 is amember of the SUT2 group (Schulze et al. (2000) FEBS Letters 485:189-194). The SUT4 group, of which AtSUT4 is a member, are low affinitytransporters which are expressed in sink tissues and may function inphloem loading within source tissues (Weise et al. (2000) The Plant Cell12:1345-1355). Active variants and fragments of sucrose transporterswill retain the ability to transport sucrose.

Others refer to sucrose transporters based on substrate affinity values.Herein, sucrose transporters are further classified based onphylogenetic tree analysis. One example of phylogenetic tree analysiscan be found in Kuhn (Kuhn, C, Plant biol (2003) 5: 215-232).

Overexpression of sucrose transporters in seeds affects seed developmentand results in increased carbon flux into developing cotyledons (Roscheet al. (2002) The Plant Journal 30(2):165-175; Rosche et al. (2005)Functional Plant Biology 32: 997-1007). In addition, studies have shownthat heterologous expression of sucrose transporters results inincreased sucrose uptake (Leggewie et al. (2003) Planta 217: 158-167).

Methods and compositions relating to the overexpression of sucrosetransporters (such as SUT2 or SUT4 sucrose transporters) in high oilplants are provided. In one embodiment, a high oil plant comprisingrecombinant DNA constructs that overexpress SUT2 or SUT4 sucrosetransporters which further increase plant seed oil production comparedto a high oil plant comprising recombinant DNA constructs that do notoverexpress SUT2 or SUT4 transporters and do not further increase plantseed oil production is provided. Overexpression of SUT2 or SUT4 sucrosetransporters in a high oil plant resulting in further increase in oilproduction provides a significant advantage in the state of the art.

It is well understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. A number of promoters can be used inrecombinant DNA constructs to overexpress sucrose transporters and/orthe sequences that influence fatty acid accumulation (i.e., DGAT, le1and ODP1 sequences) in plants. In particular embodiments of theinvention, these promoters include, but are not limited to, the Glycinemax annexin promoter (Kinney and Liu, U.S. Pat. No. 7,129,089), theGlycine max glycinin Gy1 promoter (WO Patent No. 2004/071467), Glycinemax β-conglycinin α′-subunit (Beachy et al., EMIBO J. 4:3047-3053(1985), Glycine max kunitz trypsin inhibitor (Jofuku et al., Plant Cell1: 1079-1093 (1989), Glycine max albumin 2S (U.S. Pat. No. 6,177,613),Pisum sativum legumin A1 (Rerie et al., Mol, Gen. Genet. 225: 148-157(1991)), Glycine max β-conglycinin β-subunit (WO 2004/071467), Glycinemax BD30 (also called P34) (U.S. Pat. No. 7,129,089), and Pisum sativumlegumin A2 (Rerie et al., Mol. Gen. Genet, 225:148-157 (1991)). Each ofthese references is herein incorporated by reference in their entirety.

A recombinant construct can further comprise a terminator sequenceoperably linked to the polynucleotide of interest. Terminators include,but are not limited to, bean phaseolin 3′ terminator (WO 2004/071467),Glycine max Myb2 3′ (U.S. application Ser. No. 12/486,793), Glycine maxkunitz trypsin inhibitor 3′ (WO 2004/071467), Glycine max BD30 (alsocalled P34) 3′ (WO 2004/071467), Pisum sativum legumin A2 3′ (WO2004/071467), and Glycine max albumin 2S 3′ (WO 2004/071467).

For instance, PCT Publication No. WO 2004/071467 and U.S. Pat. No.7,129,089 describe the isolation of a number of promoter andtranscription terminator sequences for use in embryo-specific expressionin soybean. Furthermore, PCT Publication Nos. WO 004/071467 and U.S.Pat. No. 7,129,089 describe the synthesis of multiplepromoter/gene/terminator cassette combinations by ligating individualpromoters, genes, and transcription terminators together in uniquecombinations. Generally, a NotI site flanked by the suitable promoter(promoters include, but are not limited to, Glycine max annexin promoter(Kinney and Liu, U.S. Pat. No. 7,129,089), the Glycine max glycinin Gy1promoter (WO Patent No. 2004/071467), Glycine max β-conglycinin α′subunit (Beachy et at, EMBO J. 4:3047-3053 (1985), Glycine max kunitztrypsin inhibitor (Jofuku et al., Plant Cell 1: 1079-1093 (1989),Glycine max albumin 2S (U.S. Pat. No. 6,177,613), Pisum sativum leguminA1 (Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991)), Glycine maxβ-conglycinin β-subunit (WO 2004/071467), Glycine max BD30 (also calledP34) (U.S. Pat. No. 7,129,089), and Pisum sativum legumin A2 (Rerie etal., Mol. Gen. Genet. 225:148-157 (1991)) and a transcription terminator(transcription terminators include, but are not limited to, beanphaseolin 3′ terminator (WO 2004/071467) Glycine max Myb2 3′ (U.S.application Ser. No. 12/486,793), Glycine max kunitz trypsin inhibitor3′ (WO 2004/071467), Glycine max BD30 (also called P34) 3′ (WO2004/071467), Pisum sativum legumin A2 3′ (WO 2004/071467), and Glycinemax albumin 2S 3′ (WO 2004/071467) is used to clone the desired gene.NotI sites can be added to a gene of interest using PCR amplificationwith oligonucleotides designed to introduce NotI sites at the 5′ and 3′ends of the gene. The resulting PCR product is then digested with NotIand cloned into a suitable promoter/NotI/terminator cassette. Althoughgene cloning into expression cassettes is often done using the NotIrestriction enzyme, one skilled in the art can appreciate that a numberof restriction enzymes can be utilized to achieve the desired cassette.Further, one skilled in the art will appreciate that other cloningtechniques including, but not limited to, PCR-based orrecombination-based techniques can be used to generate suitableexpression cassettes.

In addition, WO 2004/071467 and U.S. Pat. No. 7,129,089 describe thefurther linking together of individual promoter/gene/transcriptionterminator cassettes in unique combinations and orientations, along withsuitable selectable marker cassettes, in order to obtain the desiredphenotypic expression. Although this is done mainly using differentrestriction enzymes sites, one skilled in the art can appreciate that anumber of techniques can be utilized to achieve the desiredpromoter/gene/transcription terminator combination or orientations. Inso doing, any combination and orientation of embryo-specificpromoter/gene/transcription terminator cassettes can be achieved. Oneskilled in the art can also appreciate that these cassettes can belocated on individual DNA fragments or on multiple fragments whereco-expression of genes is the outcome of co-transformation of multipleDNA fragments.

It is recognized that other promoters may be used in recombinant DNAconstructs to over-express sucrose transporters and/or otherpolypeptides of the invention in plants. Such promoters can be thenative promoter of the polynucleotides (i.e., to the sucrosetransporters or to the sequence whose expression influence oilaccumulation, DGAT, Lec1 or ODP-1), or they may be selected based on thedesired outcome. Thus, nucleic acids of the invention can be combinedwith constitutive, tissue-preferred, or other promoters for expressionin plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference.

Compositions provided include plants, plant cells, and plant seedshaving incorporated into their genomes a first recombinant orheterologous DNA construct and a second recombinant or heterologous DNAconstruct. The first DNA construct comprises a first polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide when expressed results in increased oil in the plant. Thesecond DNA construct comprises a second polynucleotide operably linkedto at least one regulatory element, wherein the second polynucleotideencodes a polypeptide that is a SUT4 or SUT2 sucrose transporter or anactive variant or fragment thereof. The plant seeds from such plantsexhibit an increased oil content when compared to a plant seed that doesnot comprise the second polynucleotide encoding the SUT4 or SUT2 sucrosetransporter or an active variant or fragment thereof.

In one embodiment, the first polynucleotide which when expressed resultsin increased oil content in the plant comprises a sequence encoding aDGAT polypeptide or an active variant or fragment thereof, including butnot limited to a sequence encoding a DGAT1 polypeptide and/or a DGAT2polypeptide and the second polynucleotide encodes a SUT4 and/or SUT2sucrose transporter. Non-limiting examples of DGAT sequences that can beused are set forth in Table 1 and Table 5 or active fragments orvariants thereof. In still further embodiments, the DGAT sequenceemployed comprises a polynucleotide encoding a polypeptide having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the DGAT sequences set forth in any one of SEQ IDNOS: 180, 182, 184, 186, 188, 190 or 191. Such sequences will continueto have DGAT activity and thus increase the oil content of a seed whenexpressed the seed or the plant. In such embodiments, the secondpolynucleotide may comprise a polynucleotide encoding a SUT2 and/or SUT4polypeptide or an active variant or fragment thereof. Non-limitingexamples of SUT2 or SUT4 polypeptide are set forth in Table 1. Thus, theSUT2 and/or SUT4 polypeptides employed can comprises a polynucleotideencoding a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the SUT2 or SUT4sequences set forth in any one of SEQ ID NOS: 4, 6, 34, 36, 38, 40-50,52-83, 85, 150, and/or 178. Such sequences will continue to have SUT2 orSUT4 activity (i.e., transport sucrose). The plant seeds from suchplants exhibit an increased oil content when compared to a plant seedthat does not comprise the second polynucleotide encoding the SUT4 orSUT2 sucrose transporter.

In a further non-limiting embodiment, the plant, plant seed, or plantcell comprises a first polynucleotide encoding a DGAT polypeptide havingat least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity sequence identity to SEQ ID NO: 180, 182, 184, 186,188, 190 or 191, and the second polynucleotide encodes a sucrosetransporter polypeptide having an amino acid sequence of at least 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitywhen compared to SEQ ID NOs: 38, 40, 34, 6, and/or 4. Such sequenceswill continue to have DGAT activity and thus increase the oil content ofa seed when expressed the seed or the plant and will continue to haveSUT2 or SUT4 activity (i.e., transport sucrose respectively.

The plant seeds from such plants exhibit an increased oil content whencompared to a plant seed that does not comprise the secondpolynucleotide encoding the SUT4 or SUT2 sucrose transporter.

Further compositions provided include plants, plant cells, and plantseeds having incorporated into their genomes a first and a secondrecombinant or heterologous DNA construct, wherein the firstpolynucleotide encodes a Lecl polypeptide or an active variant orfragment thereof, and the second polynucleotide encodes a SIM and/or aSUT4 polypeptide or an active variant or fragment thereof, including,but not limited to, the SUT4 and SUT2 polypeptides disclosed in Table 1.Thus, in specific embodiments, the SUT2 and/or SUT4 polypeptidesemployed can comprises a polynucleotide encoding a polypeptide having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the SUT2 or SUT4 sequences set forth in any one ofSEQ 1D NOS: 4, 6, 34, 36, 38, 40-50, 52-83, 85, 150, and/or 178. Theplant seeds from such plants exhibit an increased oil content whencompared to a plant seed that does not comprise the secondpolynucleotide encoding the SUT4 OF SUT2 sucrose transporter. Suchsequences will continue to have SUT2 or SUT4 activity (i.e., transportsucrose).

Further compositions provided include plants, plant cells, and plantseeds having incorporated into their genomes a first and a secondrecombinant or heterologous DNA construct, wherein the firstpolynucleotide comprises a polynucleotide encoding an ODP-1 polypeptideor an active variant or fragment thereof, and the second polynucleotideencodes a SUT2 and/or a SUT4 polypeptide or active variant or fragmentthereof, including, but not limited to, the SUT4 and SUT2 polypeptidesdisclosed in Table 1. Thus, in specific embodiments, the SUT2 and/orSUT4 polypeptides employed can comprises a polynucleotide encoding apolypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the SUT2 or SUT4 sequences setforth in any one of SEQ ID NOS: 4, 6, 34, 36, 38, 40-50, 52-83, 85, 150,and/or 178. Such sequences will continue to have SUT2 or SUT4 activity(i.e., transport sucrose). The plant seeds from such plants exhibit anincreased oil content when compared to a plant seed that does notcomprise the second polynucleotide encoding the SUT4 or SUT2 sucrosetransporter.

Still further compositions provided include plants, plant cells, andplant seeds having incorporated into their genomes a first and a secondrecombinant or heterologous DNA construct, wherein the firstpolynucleotide, when expressed result in silencing of an expressed generesulting in fatty acid accumulation, including but not limited to, thesilencing of phosphoglucomutase, and the second polynucleotide comprisesa SUT2 and/or a SUT4 polypeptide or an active variant or fragmentthereof, including but not limited to those disclosed in Table 1. Theplant seeds from such plants exhibit an increased oil content whencompared to a plant seed that does not comprise the secondpolynucleotide encoding the SUT4 or SUT2 sucrose transporter.

As discussed elsewhere herein, any of the plants, plant cells or seedsdisclosed herein can be from any plant, including, but not limited to,maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton,rice, barley, millet, sugar cane or switchgrass.

Methods are provided for further increasing oil content in a high oilplant seed. The Methods comprises (a) introducing into a regenerablehigh oil plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence,wherein the polynucleotide encodes a polypeptide that is a SUT4 and/orSUT2 sucrose transporter polypeptide or an active variant or fragmentthereof; (b) regenerating a transgenic plant from the regenerable plantcell, where the transgenic plant comprises in its genome the recombinantDNA construct; (c) obtaining a progeny plant derived the transgenicplant, where the progeny plant comprises in its genome the recombinantDNA construct and exhibits increased oil content when compared to aplant not comprising the recombinant DNA construct.

In such methods, any polynucleotide encoding a SUT2 and/or SUT4polypeptide set forth in Table 1, or active variants or fragmentsthereof can be employed. For example, in one embodiment, the recombinantDNA construct encodes a polypeptide having an amino acid sequence of atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, or 99%sequence identity, when compared to SEQ ID NOs: 38, 40, 34, 6, 4, 36,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52-83, 85, 150, or 178, whereinsaid sequence continue to encode a polypeptide having sucrosetransporter activity.

In one embodiment, the regenerable high oil plant cell employed in themethod comprises a second recombinant DNA construct comprising asequence that influences fatty acid accumulation, such as, a sequenceencoding a diacylglycerol acyltransferase (DGAT) polypeptide or anactive variant or fragment thereof, a Lec1 transcription factorpolypeptide or an active variant or fragment thereof or an ODP-1transcription factor polypeptide or an active variant or fragmentthereof.

In one non-limiting method, the regenerable high oil plant cell employedin the method has stably incorporated into its genome a recombinant orheterologous construct comprising a DGAT polypeptide or an activevariant or fragment thereof, including but not limited to a sequenceencoding a DGAT1 polypeptide or a DGAT2 polypeptide. Non-limitingexamples of DGAT sequences that can be present in the regenerable highoil plant cell include those set forth in Table 1 and Table 5 or anactive fragments or variants thereof in still further embodiments, theDGAT sequence employed comprises a polynucleotide encoding a polypeptidehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% sequence identity to the DGAT sequences set forth in any one ofSEQ ID NOS: 180, 182, 184, 186, 188, 190, or 191. Such sequences willcontinue to have DGAT activity and thus increase the oil content of aseed when expressed the seed or the plant.

Further provided is a method of evaluating increased oil content in aplant seed. The method comprises (a) obtaining a transgenic plant,wherein the transgenic plant comprises in its genome a recombinant orheterologous DNA construct comprising a polynucleotide operably linkedto at least one regulatory element, wherein the polynucleotide encodes aSUT4 or SUT2 polypeptide or an active variant or fragment thereof; (b)obtaining a progeny plant derived from the transgenic plant, where theprogeny plant comprises in its genome the recombinant DNA construct; (c)obtaining seed from the progeny plant; and (d) evaluating the seed forincreased oil content compared to a plant seed not comprising therecombinant or heterologous DNA construct.

In such methods, any polynucleotide encoding a SUT2 and/or SUT4polypeptide set forth in Table 1, or active variants or fragmentsthereof can be employed, For example, in one embodiment, the recombinantDNA construct encodes a polypeptide having an amino acid sequence of atleast 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity, when compared to SEQ ID NOs: 38, 40, 34, 6, 4, 36,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52-83, 85, 150, or 178, whereinsaid sequence continue to encode a polypeptide having sucrosetransporter activity.

In one embodiment, the transgenic plant employed in the method canfurther comprise a second recombinant or heterologous DNA constructencoding a diacylglycerol acyltransferase (DGAT) polypeptide or anactive variant or fragment thereof, a Lec1 transcription factorpolypeptide or an active variant or fragment thereof, or an ODP-1transcription factor polypeptide or an active variant or fragmentthereof.

In one non-limiting method, the transgenic plant employed in the methodhas stably incorporated into its genome a recombinant or heterologousconstruct comprising a DGAT polypeptide or an active variant or fragmentthereof, including but not limited to a sequence encoding a DGAT1polypeptide or a DGAT2 polypeptide. Non-limiting examples of DGATsequences that can be present in the regenerable high oil plant cellinclude those set forth in Table 1 and Table 5 or an active fragment orvariant thereof. In still further embodiments, the DGAT sequenceemployed comprises a polynucleotide encoding a polypeptide having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to DGAT sequences set forth in any one of SEQ ID NOS:180, 182, 184, 186, 188, 190, or 191.

As discussed elsewhere herein, any of the methods disclosed herein canemploy any plant, including, but not limited to, maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, sugar cane or switchgrass.

Further provide are isolated polynucleotides and polypeptides.Compositions include an an isolated or recombinant polynucleotidecomprising: (a) a nucleotide sequence encoding a polypeptide having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NOs: 52, 85, 150, or 178, sequenceswill continue to have sucrose transport activity; or, (b) the complementof the nucleotide sequence, wherein the complement and the nucleotidesequence contain the same number of nucleotides and are 100%complementary. Further provided is an isolated or recombinantpolynucleotide which comprising at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity the nucleotidesequence of SEQ ID NOs: 51, 84, 149, or 177.

Further provided is a recombinant DNA construct comprising (a) anucleotide sequence encoding a polypeptide having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 52, 85, 150, or 178; (b) the complement of thenucleotide sequence, wherein the complement and the nucleotide sequencecontain the same number of nucleotides and are 100% complementary; or(c) a nucleotide sequence comprising at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thenucleotide sequence of SEQ ID NOs: 51, 84, 149, or 177; wherein thenucleotide sequence is operably linked to at least one regulatorysequence.

Additional compositions include cells, plants, plant cells, and seedcomprising a heterologous polynucleotide comprising (a) a nucleotidesequence encoding a polypeptide, having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NOs: 52, 85, 150, or 178, wherein the sequences will continue to havesucrose transport activity; (b) the complement of the nucleotidesequence, wherein the complement and the nucleotide sequence contain thesame number of nucleotides and are 100% complementary; or (c) anucleotide sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity the nucleotidesequence of SEQ ID NOs: 51, 84, 149, or 177, wherein said sequenceencode a polypeptide having sucrose transport activity. In furtherembodiments, the heterologous polynucleotide can be in a recombinant DNAconstruct. As discussed elsewhere herein, the cell may be eukaryotic,e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterium.In another embodiment, the plants or seeds derived from said plantwherein the plant is selected from, but not limited to, the groupconsisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.

Further provide are isolated or recombinant polypeptides which comprisean amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:52, 85, 150, or 178.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Furthermore, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Cloning of Arabidopsis thaliana Sucrose Transporter Genes

This example describes cloning of Arabidopsis thaliana sucrosetransporter genes, ATSUC2 (AT1G227110, SEQ ID NO: 1), ATSUT2 (AT2G02860,SEQ ID NO: 3), and ATSUT4 (AT1G09960, SEQ ID NO: 5). Total RNA wasprepared from 1 month-old Arabidopsis thaliana seedlings using TRIzol®Reagent (Invitrogen™, USA) following the manufacturer's protocol. Tomake first strand cDNA, a reverse transcription reaction was carried outas follows. A mixture (1 μL of 50 μM dTVN, 1 μL dNTP mix (10 μM each), 5μg of total RNA, and 10 μL of water) was heated at 65° C. for 5 min andimmediately placed on ice for 1 min. The reaction mixture wassupplemented with 4 μL of 5× reverse transcription buffer, 1 μL of 0.1 MDTT, 1 82 L of anti RNase, and SuperScript III (Invitrogen™, USA). Thereverse transcription reaction was carried out for 1 hat 50° C. Thereaction was stopped by incubating at 70° C. for 15 min. Twentymicroliters of water was added to the reaction. Synthesized first strandcDNAs were used as templates in subsequent PCR reactions.

The PCR reactions, with the first-strand Arabidopsis thaliana cDNAs astemplate, were individually carried out in 50 μL total volumecomprising: 1 μL each of 10 μM forward and reverse primers, 2 μL cDNAs,5 μL 10× PCR buffer, 1 μL dNTP mix (10 μM each), 39 μL water and 1 μLExpand polymerase (Roche Applied Science, Indianapolis, Ind.). Primersdesigned and used to introduce a NotI site flanking the gene were YOL237(SEQ ID NO: 7) and YOL132 (SEQ ID NO: 8) for ATSUC2, YOL174 (SEQ ID NO:9) and YOL175 (SEQ ID NO: 10) for ATSUT2, and YOL172 (SEQ ID NO: 11) andYOL173 (SEQ ID NO: 12) for ATSUT4. Amplification was carried out at 94°C. for 3 min, followed by 30 cycles at 94° C. for 30 sec, 58° C. for 30sec, and 72° C. for 2 min, followed by a final elongation cycle at 72°C. for 6 min. PCR products were gel-purified, cloned into pCRR2.1(Invitrogen™, USA) using manufacturer instructions and weresequence-verified. The resulting plasmids for each gene were set forthas ATSUC2-pCRR2.1 (SEQ ID NO: 13), ATSUT2-pCR2.1 (SEQ ID NO: 14), andATSUT4-pCRR2.1 (SEQ ID NO: 15) respectively.

To aid protein analyses, ATSUC2, ATSUT2, or ATSUT4 with a hexa-histidinetag at the carboxyl terminus (SEQ ID NO: 16; 17; and 18, respectively)was created by PCR reactions as follows. Oligonucleotide primers usedwere YOL412 (SEQ ID NO: 19) and YOL413 (SEQ ID NO: 20) for ATSUC2,YOL416 (SEQ ID NO: 21) and YOL417 (SEQ ID NO: 22) for ATSUT2, and YOL414(SEQ ID NO: 23) and YOL415 (SEQ ID NO: 24) for ATSUT4. Primers weredesigned to have a Nod restriction site flanking the gene. Templateswere respective plasmids carrying the identical sucrose transportergenes either as in ATSUC2 (AT1G22710, SEQ ID NO: 1), ATSUT2 (AT2G02860,SEQ ID NO: 3), or ATSUT4 (AT1G09960, SEQ ID NO: 5). PCR reaction wascarried out in a 50 μL total volume comprising: 1 μL each of 10 μMrespective primers, 1 μL template DNA (100 ng), 10 μL 5× PCR buffer, 1μL dNTP mix (10 μM each), 36 μL water and 0.5 μL Phusion polymerase (NewEngland Biolabs, Inc., Ipswich, Mass.). Amplification was carried out at98° C. for 30 sec, followed by 30 cycles at 98° C. for 10 sec, 55° C.for 15 sec, and 72° C. for 30 sec, followed by a final elongation cycleat 72° C. for 6 min. PCR products were gel-purified and cloned intopCRR4BLUNT-TOPOR (Invitrogen) using manufacturer instructions. Based onsequencing analyses, the plasmids with the consensus sequences were setforth as RTW155 (ATSUC2HIS6) (SEQ ID NO: 25), RTW156 (ATSUT4HIS6) (SEQID NO: 26), and RTW157 (ATSUT2HIS6) (SEQ ID NO: 27).

Example 2 Functional Assays of Arabidopsis thaliana Sucrose TransporterGenes in Saccharomyces cerevisiae

This example describes functional activity assays of Arabidopsisthaliana sucrose transporters in an invertase-deleted yeast strain (seeon the World Wide Web athttp://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html).The method used was based on Sauer and Stolz (Plant J. (1994) 6: 67-77)with modification.

Using the yeast expression vector, pY75 (U.S. application Ser. No.12/126,161 (SEQ ID NO: 3)), yeast expression plasmids containingArabidopsis thaliana sucrose transporters described in Example 1 wereconstructed and employed in assays after transformation into yeast.

Construction of RTW247 (Yeast ATSUC2HIS):

The construction of plasmid RTW155 (ATSUC2HIS6) is described inExample 1. A fragment containing ATSUC2HIS6 gene was excised from RTW155with NotI restriction enzyme digestion. This DNA was ligated to NotIlinearized, dephosphorylated pY75 vector DNA to give RTW247 (yeastATSUC2HIS) (SEQ ID NO: 28).

Construction of RTW248 (Yeast ATSUT4HIS):

The construction of plasmid RTW156 (ATSUT4HIS6) is described inExample 1. A fragment containing ATSUT4HIS6 gene was excised from RTW156with NotI restriction enzyme digestion. This DNA was ligated to NotIlinearized, dephosphorylated pY75 vector DNA to give RTW248 (yeastATSUT4HIS) (SEQ ID NO: 29).

Construction of RTW249 (Yeast ATSUT2HIS):

The construction of plasmid RTW157 (ATSUT2HIS6) is described inExample 1. A fragment containing ATSUT2HIS6 gene was excised from RTW157with NotI restriction enzyme digestion. This DNA was ligated to NotIlinearized, dephosphorylated pY75 vector DNA to give RTW249 (yeastATSUT2HIS) (SEQ ID NO: 30).

Construction of RTW250 (Yeast ATSUC2):

The construction of plasmid ATSUC2-pCRR2.1 is described in Example 1. Afragment containing ATSUC2 gene was excised from ATSUC2-pCRR2.1 withNotI restriction enzyme digestion. This DNA was ligated to NotIlinearized, dephosphorylated pY75 vector DNA to give RTW250 (yeastATSUC2) (SEQ ID NO: 31).

Construction of RTW251 (Yeast ATSUT4):

The construction of plasmid ATSUT4-pCRR2.1 is described in Example 1. Afragment containing ATSUT4 gene was excised from ATSUT4-pCRR2.1 withNotI restriction enzyme digestion. This DNA was ligated to NotIlinearized, dephosphorylated pY75 vector DNA to give RTW251 (yeastATSUT4) (SEQ ID NO: 32).

Transformation of Yeast

Plasmids RTW247 (yeast ATSUC2HIS), RTW248 (yeast ATSUT4HIS), RTW249(yeast ATSUT2HIS), RTW250 (yeast ATSUC2), RTW251 (yeast ATSUT4), and theempty pY75 vector were transformed into the Saccharomyces cerevisiaestrain that has deletion in invertase gene (see on the World Wide Web athttp://www.sequence.stanford.edu/group/yeast_deletion_project/deletions3.html)using S. c. EasyComp Transformation Kit (Invitrogen) and manufacturer'sinstruction. Recombinant yeast colonies were selected on DOB agar platessupplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). DOB media arecomposed of 1.7 g yeast nitrogen base, 5 g ammonium sulfate, and 20 gDextrose per liter.

Sucrose Transporter Assays in Yeast

For functional assays of sucrose transporters in yeast, single colonyfrom each transformation was inoculated in 10 mL of DOB mediasupplemented with CSM-leu and grown at 30° C. for 48 h. This seedculture was used to inoculate the second culture of 40 mL DOB mediasupplemented with CSM-leu. The culture was incubated at 30° C. for 16 h.After checking cell density at OD575, cells were harvested bycentrifugation. Cells were washed by resuspending in culture mediacomposed with DOB supplemented with CSM-leu and 20 mM sucrose, followedby centrifugation. This step was performed twice. After washing, 100 μLof an OD575 culture of cells was added to the final volume of 100 ml ofDOB supplemented with CSM-leu and 20 mM sucrose.

Three replicates of each yeast culture were harvested by centrifugation(2,000×g for 10 min) at 1, 2, 3, 4 and 6 hours after culture initiation.The media was discarded and the pelleted cells were re-suspended in 10mL of deionized water, to remove any residual media. The cells werepelleted again, the supernatant discarded, and the pellets re-suspendedin 1 mL deionized water prior to quantitative transfer to pre-weighed 2mL capacity centrifuge tubes. The samples were frozen in liquid nitrogenand stored at −80° C. prior to lyophilization. The dry samples werere-weighed and the dry weight of the cell pellets (average ˜3mg/culture) was calculated and used to normalize the carbohydrateconcentrations (see below).

The dried yeast pellets were suspended in 1 mL 80% aqueous ethanol and,with the tubes in ice, sonicated for 3×30 sec at a 50% power settingusing a Vibra-Cell fitted with a Model ASI probe (Sonics & MaterialsInc; Newtown, Conn., USA). The cell debris was pelleted bycentrifugation (16,000×g for 10 min) and the supernatants weretransferred to clean 13×100 mm screw-capped glass tubes fitted withTeflon® lined closures. The pellets were extracted 3 more times with 1mL volumes of 80% ethanol as follows. After addition of the ethanol thetubes were vortex mixed and placed into an ultrasonic bath (VWRScientific Model 750D) filled with water heated to 60° C. The sampleswere sonicated at full-power (˜360 W) for 15 min and were thencentrifuged (10 min×16,000×g) with the supernatant from each extractpooled with those from the previous extracts. Internal standard (10 μL,β-phenyl giucopyranoside (Sigma-Aldrich P6876); 0.5000+/0.0010 g/100 mLstock in water) was added to each pooled supernatant prior to drying ina Speedvac.

The dried samples were solubilized in anhydrous pyridine (Sigma-AldrichP57506) containing 30 mg/mL of hydroxylamine HCl (Sigma-Aldrich 159417).Samples were placed on an orbital shaker (300 rpm) overnight and werethen heated for 1 hr (75° C.) with vigorous vortex mixing applied every15 min. After cooling to room temperature, 1 mL hexamethyldisilazane(Sigma-Aldrich H-4875) and 100 μL trifluoroacetic acid (Sigma-AldrichT6508) were added. The samples were vortex mixed and the precipitateswere allowed to settle prior to transferring the supernatants to GCsample vials.

Samples were analyzed on an Agilent 6890 gas chromatograph fitted with aDB-17MS capillary column (15 m×0.32 mm×0.25 μm film). Inlet and detectortemperatures were both 275° C. After injection (2 μL, 20:1 split) theinitial column temperature (150° C.) was increased to 180° C. at a rateof 3° C./min and then at 25° C./min to a final temperature of 320° C.The final temperature was maintained for 10 min. The carrier gas was H₂at a linear velocity of 51 cm/sec. Detection was by flame ionization.Data analysis was performed using Agilent ChemStation software. Sucrosewas quantified relative to the internal standard and detector responseswere applied (calculated from standards run with each set of samples).Final carbohydrate concentrations were expressed on a tissue dry weightbasis.

FIGS. 1-3 present sucrose levels recovered from yeast cells aftervarious times in culture for SUT2 (FIG. 1), SUT4 (FIG. 2), and SUC2(FIG. 3).

For all three sucrose transporter classes tested, increases inintracellular sucrose levels were observed as the time in cultureincreased. Yeast cells carrying the empty pY75 vector were used as thecontrol and showed no sucrose accumulation even after 6 hours inculture;

growth of the control yeast, as assessed by the dry cell mass at eachharvest time, was at least equivalent to the cells expressing thevarious transporters. All three classes of sucrose transporter retainedtheir capacity to transport sucrose even with the HIS-Tag fused to thecarboxy terminus of the protein.

The cloned genes result in the expression of proteins with the apparentcapacity to transport sucrose and the proteins retain this ability evenwhen fused with a C-terminal HIS-tag.

Example 3 Identifying Sucrose Transporter Gene Homologs in Soy

Soybean homologs of the Arabidopsis Sut2 (SEQ II) NO: 4) and Sut4 (SEQID NO: 6) genes were identified by conducting BLAST (Basic LocalAlignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410(1993)) searches for similarity to sequences contained in the SoybeanGenome Project, DoE Joint Genome Institute “Glyma1.01” gene set.Specifically, the TBLASTN algorithm provided by National Center forBiotechnology Information (NMI) was used with default parameters exceptthe Filter Option was set to OFF.

In this way, two soy putative cDNA sequences were identified withhomology to Arabidopsis Sut2 protein (Glyma08g40980 (SEQ ID NO: 33) andGlyma18g15950 (SEQ ID NC): 35)) and two soy putative cDNA sequences wereidentified with homology to Arabidopsis Sut4 (Glyma02g38300 (SEQ ID NO:37) and Glyma04g09460 (SEQ ID NO: 39)).

The SEQ ID NOs for DNA CDS and amino acid sequences of each of the soySut homologs as well as the percent identity to the correspondingArabidopsis proteins are shown in Table 2.

Sequence percent identity calculations performed by the Clustal V method(Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci. 5:151-153(1989); Higgins et al., Comput. Appl. Biosci. 8:189-191 (1992)) weredone using the MegAlign™v6.1 program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) with the defaultparameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5 and GAP LENGTH PENALTY=10).

TABLE 2 Soy homologs to the Arabidopsis Sut2 and Sut4 genes NucleotideAmino Acid % Sut Gene Soy Sut (SEQ (SEQ Amino Acid Subfamily Homolog IDNO:) ID NO:) Identity Sut2 Glyma08g40980 33 34 68.4% Sut2 Glyma18g1595035 36 66.4% Sut4 Glyma02g38300 37 38 66.0% Sut4 Glyma04g09460 39 4063.9%

Example 4 Identification of Sucrose Transporter Homologs

Plant homologs of Arabidopsis thaliana SUT1, SUT2 and SUT4 homologs wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also theexplanation of the BLAST algorithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health) searches for similarityto amino acid sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The DNA sequences from clones can be translated in allreading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States, Nat. Genet. 3:266-272 (1993)) provided bythe NCBI. Alternatively, the polypeptides encoded by the cDNA sequencescan be analyzed for similarity to all publicly available amino acidsequences contained in the “nr” database using the BLASTP algorithmprovided by the National Center for Biotechnology Information (NCBI).For convenience, the P-value (probability) or the E-value (expectation)of observing a match of a cDNA-encoded sequence to a sequence containedin the searched databases merely by chance as calculated by BLAST arereported herein as “pLog” values, which represent the negative of thelogarithm of the reported P-value or E-value. Accordingly, the greaterthe pLog value, the greater the likelihood that the cDNA-encodedsequence and the BLAST “hit” represent homologous proteins.

EST sequences can be compared to the GenBank database as describedabove. ESTs that contain sequences more 5- or 3-prime can be found byusing the BLASTN algorithm (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)) against the DUPONT proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described above.

Homologous genes belonging to different species can be found bycomparing the amino acid sequence of a known gene (from either aproprietary source or a public database) against an EST database usingthe tBLASTn algorithm. The tBLASTn algorithm searches an amino acidquery against a nucleotide database that is translated in all 6 readingframes. This search allows for differences in nucleotide codon usagebetween different species, and for codon degeneracy.

Table 3 lists Arabidopsis thaliana homologs that are described herein,the corresponding identifier (SEQ ID NO) as used in the attachedSequence Listing, the SUT subfamily designation, and species. Sucrosetransporters in Table 3 were classified based on the phylogenetic treefrom the review article by Kuhn (Kuhn, C, Plant biol (2003) 5: 215-232).

TABLE 3 SUCROSE TRANSPORTER HOMOLOGS SUT Sequence Description SEQ Family(NCBI GI NO:) Species ID NO. SUT2 AtSUT2 (At2G02860; NCBI GI NO.Arabidopsis 4 973404) thaliana Glyma08g40980.1 Glycine max 34Glyma18g15950.1 Glycine max 36 CsSUT2 (NCBI GI NO. 21063927) Citrussinensis 41 EuSUT2 (NCBI GI NO. 61657989) Eucommia ulmoides 42 StSUT2(NCBI GI NO. 31096339) Solanum tuberosum 43 LeSUT2 (NCBI GI NO.10119908) Lycopersicon 44 esculentum LOC_Os02g58080_SUC3 Oryza sativa 45cfp5n.pk008.k9_fis Zea mays 46 Sb04g038030.1 Sorghum bicolor 47 PmSUT2(NCBI GI NO. 31455370) Plantago major 48 MeSUT2 (NCBI GI NO. 74476789)Manihot esculenta 49 HbSUT2 (NCBI GI NO. 116008244) Hevea brasiliensis50 Pn_Node_9230 Paspalum 52 notatum ZmSUT1 (NCBI GI NO. 162463612) Zeamays 53 Sb01g045720.1 Sorghum bicolor 54 cepe7.pk0015.d10 Zea mays 55TaSUT1A (NCBI GI NO. 20152871) Triticum aestivum 56 TaSUT1b (NCBI GI NO.20152873) Triticum aestivum 57 TaSUT1D (NCBI GI NO. 19548165) Triticumaestivum 58 HvSUT1 (NCBI GI NO. 71890897) Hordeum vulgare 59LOC_Os03g07480 Oryza sativa 60 LOC_Os10g26470_SUC1 Oryza sativa 61Sb01g022430.1 Sorghum bicolor 62 cfp1n.pk007.b22_fis Zea mays 63LOC_Os02g36700_BoSUT1 Oryza sativa 64 Sb04g023860.1 Sorghum bicolor 65Sb07g028120.1 Sorghum bicolor 66 BoSUT1(NCBI GI NO. 66269698) Bambusaoldhamii 67 cfp1n.pk065.p4_fis Zea mays 68 cfp3n.pk071.b8_fis Zea mays69 Pn_Node_40538 Paspalum notatum 150 SUT4 ATSUT4 (At1G09960; NCBI GINO. Arabidopsis 6 15218362) thaliana Glyma02g38300.1 Glycine max 38Glyma04g09460.1 Glycine max 40 PsSUF4 (NCBI GI NO. 78192243) Pisumsativum 70 HbSUT5 (NCBI GI NO. 118132673) Hevea brasiliensis 71 HbSUT4(NCBI GI NO. 118132677) Hevea brasiliensis 72 MeSUT4 (NCBI GI NO.74476785) Manihot esculenta 73 VvSUC11 (NCBI GI NO. 6434829) Vitisvinifera 74 StSUT4 (NCBI GI NO. 160425326) Solanum tuberosum 75 DcSUT1aDaucus carota 76 ZmSUT4 (NCBI GI NO. 47571319) Zea mays 77 Sb08g023310.1Sorghum bicolor 78 LOG_Os12g44380_SUC4_orSUC2 Oryza sativa 79 HvSUT2(NCBI GI NO. 7024413) Hordeum vulgare 80 MdSUT4 (NCBI GI NO. 38327323)Malus domestica 81 DgSUT4 (NCBI GI NO. 49609488) Datisca glomerata 82LjSUT4 (NCBI GI NO. 28172870) Lotus japonicus 83 Pn_Node_3980 Paspalumnotatum 85 hso1c.pk009.16:fis Helianthus annuus 178 SUT1 ATSUC8(At2G14670; NCBI GI NO. Arabidopsis 86 15225986) thaliana ATSUC7(At1G66570; NCBI GI NO. Arabidopsis 87 115646796) thaliana ATSUC6(At5G43610; NCBI GI NO. Arabidopsis 88 15239921) thaliana ATSUC9(At5G06170; NCBI GI NO. Arabidopsis 89 15239949) thaliana AtSUC2(At1G22710; NCBI GI NO. Arabidopsis 2 15219938) thaliana AtSUC1(At1G71880; NCBI GI NO. Arabidopsis 90 56550707) thaliana ATSUC5(At1G71890; NCBI GI NO. 91 15217602) Glyma02g08250.1 Glycine max 92se5.pk0033.f9_Glyma16g27320.1 Glycine max 93 PvSUT1 (NCBI GI NO.78192247) Phaseolus vulgaris 94 sfl1.pk0001.g1_Glyma16g27340_&_27330Glycine max 95 PvSUT3 (NCBI GI NO. 78192251 Phaseolus vulgaris 96sgc7c.pk001.n22_Glyma02g08260.1 Glycine max 97sls2c.pk003.p4_Glyma16g27350.1 Glycine max 98 PvSUF1 (NCBI GI NO.125625363) Phaseolus vulgaris 99 SUF1_Ps (NCBI GI NO. 78192245) Pisumsativum 100 sfl1.pk0043.c7_Glyma10g36200.1 Glycine max 101 PsSUT1 (NCBIGI NO. 5230818) Pisum sativum 102 VfSut1 Vicia faba 103 HbSUT1 (NCBI GINO. 116008246) Hevea brasiliensis 104 HbSUT6 (NCBI GI NO. 167859950)Hevea brasiliensis 105 HbSUT3 (NCBI GI NO. 118132675) Hevea brasiliensis106 RcScr1 (NCBI GI NO. 468562) Ricinus communis 107 PtSUT1 (NCBI GI NO.77153413) Populus tremula × 108 Populus tremuloides EeSUT1 (NCBI GI NO.7649151) Euphorbia esula 109 hss1c.pk009.b12_fis Helianthus annuus 110vs1n.pk016.e12_fis Vernonia 111 mespilifolia VvSUT27 (NCBI GI NO.6434833) Vitis vinifera 112 AmSUT1 (NCBI GI NO. 17447420) Alonsoa 113meridionalis AbSUT1 (NCBI GI NO. 6120115) Asarina barclaiana 114 NtSUT1a(NCBI GI NO. 575351) Nicotiana tabacum 115 StSUT1 (NCBI GI NO. 439294)Solanum tuberosum 116 AgSUT2A (NCBI GI NO. 5566434) Apium graveolens 117AgSUT1 (NCBI GI NO. 4091891) Apium graveolens 118 DcSUT2 (NCBI GI NO.2969884) Daucus carota 119 BvSUT1 (NCBI GI NO. 5823000) Beta vulgaris120 SoS21 (NCBI GI NO. 549000) Spinacia oleracea 121 CsSUT1 (NCBI GI NO.21063921) Citrus sinensis 122 BoSUC2 (NCBI GI NO. 18091781) Brassicaoleracea 123 BoSUC1 (NCBI GI NO. 18091779) Brassica oleracea 124 PmSUC1(NCBI GI NO. 667047) Plantago major 125 NtSUT3 (NCBI GI NO. 4960089)Nicotiana tabacum 126

FIG. 4A-G presents an alignment of the amino acid sequences set forth inthe SUT2 family and includes SEQ ID NOs: 4, 34, 36, 41-50, 52, and 150.FIG. 5A-F presents an alignment of the amino acid sequences set forth inthe SUT2 family and includes SEQ ID NOs: 53-69. FIG. 6A-F presents analignment of the amino acid sequences set forth in the SUT4 family andincludes SEQ ID NOs: 6, 38, 40, 70-83, and 85. FIG. 7 is a chart of thepercent sequence identity and the divergence values for each pair ofamino acids sequences presented in FIG. 4A-G. FIG. 8 is a chart of thepercent sequence identity and the divergence values for each pair ofamino acids sequences presented in FIG. 5A-F. FIG. 9 is a chart of thepercent sequence identity and the divergence values for each pair ofamino acids sequences presented in FIG. 6A-F.

Sequence alignments and percent identity calculations were performedusing the MEGALIGN® program of the Li SERGENE® bioinformatics computingsuite (DNASTAR® Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal W method of alignment(Thompson, J. D., et al. (1994). Nucleic Acids Research, 22: 4673-80)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=0.2,DELAY DEVERGENT SEQS (%)=30%, DNA TRANSITION WEIGHT=0.5, PROTEIN WEIGHTMATRIX “Gonnet Series”),

Default parameters for pairwise alignments using the Clustal method wereSLOW-ACCURATE, GAP PENALTY=10, GAP LENGTH=0.10, PROTEIN WEIGHT MATRIX“Gonnet 250”

Sucrose transporter homologs were also identified from an exotic plantspecies, Paspalum notatum, commonly called Bahia grass and are includedin Table 3. One SUT4 homolog, Pn_Node_3980, was identified from Bahiagrass (SEQ ID NO: 85) and two SUT2 homologs, Pn_Node_9230 (SEQ ID NO:52) and Pn_Node_40538 (SEQ ID NO: 150), were also identified from Bahiagrass. Mining of homologs from Bahia grass was perfoiined by performinga TblastN of the Arabidopsis Sut2 and Sut4 genes, and the identified(putative) maize Sut2 and Sut4 homologs against the Bahia assemblies.The resulting hits were translated based on the blast alignments; andthe translations were aligned with the other known sucrose transporters.In cases where the Bahia assemblies were in fragments, the percentidentity to the maize genes was used to infer which Bahia fragmentsrepresented a single gene. The fragments thought to belong together werecomputationally assembled such that a translation would return thecorrect protein in a single frame. These computer assemblies were thenaligned with the other transporters as above.

Example 5 Phylogenetic Analysis of SUT1, SUT2, and SUT4

FIG. 10 present phylogenetic analysis of the SUT1, SUT2, and SUT4Arabidopsis thaliana genes and their homologs (Table 3). FIG. 11A-C ispresented as an enlargement of FIG. 10. Phylogenetic analysis andphlylogenetic tree construction were performed using Megalign ClustalWalignments exported in the MSF format. This MSF format was imported intoJalView (www.jalview.org Clamp, M., Cuff, J., Searle, S. M. and Barton,G. J. (2004). “The Jalview Java Alignment Editor”, Bioinformatics, 20,426-7). Trees were built using one of the four available methods in JalView, and the alignments were sorted based on the resulting tree. Treesand alignments were evaluated for quality and accuracy. The tree in FIG.10 was calculated with Average Distance Measure using the BLOSUM62matrix.

Example 6 Soybean Expression Vectors for Co-Expression of SucroseTransporter and DGAT Genes

In addition to the genes, promoters, terminators and gene cassettesdescribed herein, one skilled in the art can appreciate that otherpromoter/gene/terminator cassette combinations can be synthesized in away similar to, but not limited to, that described herein for theco-expression of sucrose transporter and DGAT genes. Similarly, it maybe desirable to co-express sucrose transporters of the present inventionor other sucrose transporter genes with DGAT genes of the presentinvention or other DGAT genes.

Sucrose transporters (such as those listed in, but not limited to, Table3) can be co-expressed with DGAT genes using techniques describedherein. DGAT genes (such as those listed in, but not limited to, Table5) can be used. NotI restriction enzyme sites flanking Sut and DGATgenes are added, Sut and DGAT genes are cloned into soybean expressionvectors behind suitable promoters and Sut and DGAT genes areco-expressed using methods described herein.

TABLE 4 SUT Genes Gene Organism DNA SEQ ID NO: AtSut4 Arabidopsisthaliana 5 Glyma02g38300 Glycine max 37 Glyma04g09460 Glycine max 39AtSut2 Arabidopsis thaliana 3 Glyma08g40980 Glycine max 33 Glyma18g15950Glycine max 35 AtSuc2 Arabidopsis thaliana 1

TABLE 5 DGAT genes Gene Organism Reference YLDGAT1 Yarrowia lipolyticaU.S. patent application No. 12/126,161 YLDGAT2 Yarrowia lipolytica U.S.Pat. No. 7,267,976 & US Patent Application Publication No. 20080295204YLDGAT2_Y326F Yarrowia lipolytica U.S. patent application No. 12/126,161YLDGAT2_Y326L Yarrowia lipolytica U.S. patent application No. 12/126,161YLDGAT2_Y326L Yarrowia lipolytica U.S. patent application No. 12/126,161TD_DGAT2A Torulospora delbrueckii U.S. patent application No. 12/470,517TD_DGAT2Acod Torulospora delbrueckii U.S. patent application No.12/470,517 TD_DGAT2B Torulospora delbrueckii U.S. patent application No.12/470,517 TD_DGAT2Bcod Torulospora delbrueckii U.S. patent applicationNo. 12/470,517 PA_DGAT2 Pichia anomala U.S. patent application No.12/470,517 PA_DGAT2cod Pichia anomala U.S. patent application No.12/470,517 DH_DGAT2 Debaryomyces hansenii U.S. patent application No.12/470,517 DH_DGAT2cod Debaryomyces hansenii U.S. patent application No.12/470,517 CZ_DGAT2 Candida zeylanoides U.S. patent application No.12/470,517 CZ_DGAT2cod Candida zeylanoides U.S. patent application No.12/470,517 LS_DGAT2 Lipomyces starkeyi U.S. patent application No.12/470,517 LS_DGAT2cod Lipomyces starkeyi U.S. patent application No.12/470,517 MC_DGAT2 Mucor circinelloides U.S. patent application No.12/470,517 MC_DGAT2cod Mucor circinelloides U.S. patent application No.12/470,517 PR_DGAT2 Phaffia rhodozyma U.S. patent application No.12/470,517 PR_DGAT2cod Phaffia rhodozyma U.S. patent application No.12/470,517 RG_DGAT2 Rhodotorula glutinis U.S. patent application No.12/470,517 RG_DGAT2cod Rhodotorula glutinis U.S. patent application No.12/470,517 MA_DGAT2 Mortierella alpina U.S. Pat. No. 7,198,937 and U.S.patent application No. 12/470,517 MA_DGAT2cod Mortierella alpina U.S.patent application No. 12/470,517 CC_DGAT2 Cryptococcus curvatus U.S.patent application No. 12/470,517 CC_DGAT2cod Cryptococcus curvatus U.S.patent application No. 12/470,517 LS_DGAT1 Lipomyces starkeyi U.S.patent application No. 12/470,517 LS_DGAT1cod Lipomyces starkeyi U.S.patent application No. 12/470,517 MA_DGAT1 Mortierella alpina U.S. Pat.No. 7,273,746 & U.S. patent application No. 12/470,517 MA_DGAT1codMortierella alpina U.S. patent application No. 12/470,517 GM-DGAT1Glycine max U.S. Pat. No. 7,524,945 & U.S. patent application No.12/470,569 GM-DGAT1-C9 Glycine max U.S. patent application No.12/470,569 GM-DGAT1-C10 Glycine max U.S. patent application No.12/470,569 GM-DGAT1-C11 Glycine max U.S. patent application No.12/470,569 GM-DGAT1- Glycine max U.S. patent application No. C9C10C1112/470,569 *each of the applications appearing in Table 5 is hereinincorporated by reference in their entirety.

Example 7 Expression of Arabidopsis thaliana Sucrose Transporter Genesin Soybean Somatic Embryos

It has been shown that YLDGAT1 (US Patent Application No. 2008/0295204A) can increase oil and oleic acid compared to null transgenic soybeanseeds when it is expressed either in soybean somatic embryos or insoybean seeds. The enhanced carbon flux into soybean embryos was tested.Over-expressed sucrose transporters led to further increase in oilcontent when co-expressed with YLDGAT1. Promoters that were used inplasmid construction for soybean embryo preferred over-expression ofArabidopsis thaliana sucrose transporter genes and the YLDGAT1 geneinclude, but are not limited to, Glycine max annexin and Glycine maxglycinin Gy1. Transcription terminators that were used in plasmidconstruction for soybean embryo preferred over-expression of Arabidopsisthaliana sucrose transporter genes and the YLDGATI gene include, but arenot limited to, bean phaseolin 3′ and Glycine max Myb2 3′.

Constructs containing either vector only (pKR268, SEQ ID NO: 130) orYLDGAT1 gene served as controls. These were compared to co-expressionconstructs that harbor different sucrose transporters along with YLDGAT1gene. These constructs were made as follows.

Construction of RTW218 (YLDGAT1)

Piasinid RTW218 (YLDGAT1) was constructed as follows. YLDGAT1 gene (SEQID NO: 127) was previously described in Publication No. 2008-0295204 A1and the contents of which are hereby incorporated by reference. Anexpression cassette which harbors the YLDGAT1 gene fused to glycinin Gy1promoter and the phaseolin terminator sequences, was excised as a 3.5 kbBamHI/SalI fragment from KS349 (SEQ ID NO: 129) which was previouslydescribed in Publication No Publication No. 2008-0295204 A1 (thecontents of which are hereby incorporated by reference). This DNAfragment was ligated to BamHI/SalI linearized, dephosphorylated pKR268vector DNA which contains an antibiotic marker cassette composed of 35Spromoter, hygromycin gene, and NOS terminator. The resultant plasmid wasset forth as RTW218 (YLDGAT1) (SEQ ID NO: 128).

Construction of RTW220 (MTSUC2HIS6 DGAT1 35Hyg)

The plasmid, RTW220 (ATSUC2HIS6 DGATI 35Hyg) was constructed in manysteps from several intermediate vectors. A vector, RTW147 (ANN-myb2 termsbf) (SEQ ID NO: 132), was used to prepare a cassette containing annexinpromoter (Kinney and. Liu, U.S. Pat. No. 7,129,089) and myb2 terminator(U.S. application Ser. No. 12/486793). RTW155 (ATSUC2HIS6) described inExample 1 was digested with NotI and a fragment containing ATSUC2HIS6gene was gel-isolated. The fragment was then ligated into NotI digested,dephosphorylated RTW147 (ANN-myb2 term sbf) to give RTW158p1 (ann suc2)(SEQ ID NO: 133). Expression cassette containing annexin promoter,ATSUC2HIS6 gene, and rnyb2 terminator was isolated from RTW158p1 (annsuc2) by SbfI digestion. This DNA was ligated into SbfI linearized,dephosphorylated RTW218 (YLDGAT1) plasmid. The resultant plasmid was setforth as RTW220 (ATSUC2HIS6 DGATI 35Hyg) (SEQ ID NO: 131).

Construction of RTW221 (ATSUT4HIS6 DGAT1 35Hyg)

The plasmid, RTW221 (ATSUT4HIS6 35Hyg) was constructed similarly as forRTW220 (ATSUC1HIS6 DGAT1 35Hyg). RTW156 (AT SUT4 HIS6) described inExample 1 was digested with NotI and a fragment containing ATSUT4HIS6was gel-isolated. The fragment was then ligated into NotI digested,dephosphorylated RTW147 (ANN-myb2 term sbt) to give RTW162p1 (ann sut4)(SEQ ID NO: 135). Expression cassette containing annexin promoter,ATSUT4HIS6 gene, and myb2 terminator was isolated from RTW162p1 (annsut4) by SbfI digestion. This DNA was ligated into SbfI linearized,dephosphorylated RTW218 (YLDGAT1) plasmid. The resultant plasmid was setforce as RTW221 (ATSUT4HIS6 DGAT1 35Hyg) (SEQ ID NO: 134).

Construction of RTW222 (ATSUT2HIS6 DGAT1 35Hyg)

The plasmid, RTW222 (ATSUT2HIS6 DGAT1 35Hyg) was constructed similarlyas for RTW220 (ATSUC1HIS6 DGAT1 35Hyg). RTW157 (AT SUT2 HIS6) describedin Example 1 was digested with NotI and a fragment containing ATSUT2HIS6was gel-isolated. The fragment was then ligated into NotI digested,dephosphorylated RTW147 (ANN-myb2 term sbf) to give RTW166p1 (ann sut2)(SEQ ID NO: 137). Expression cassette containing annexin promoter,ATSUT4HIS6 gene, and myb2 terminator was isolated from RTW166p1 (annsut2) by SbfI digestion. This DNA was ligated into SbfI linearized,dephosphorylated RTW218 (YLDGAT1) plasmid. The resultant plasmid was setforth as RTW222 (ATSUT2HIS6 DGAT1 35Hyg) (SEQ ID NO: 136).

Transformation and Regeneration of Soybean (Glycine max)

Transgenic soybean lines are generated by the method of particle gunbombardment (Klein et al., Nature (London) 327:70-73 (1987); U.S. Pat.No. 4,945,050) using a BIORAD Biolistic PDS1000/He instrument and eitherplasmid or fragment DNA. The following stock solutions and media areused for transformation and regeneration of soybean plants:

Stock Solutions: Sulfate 100× Stock:

37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g ZnSO₄.7H₂O, 0.0025 gCuSO₄.5H₂O Halides 100× Stock:

30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g CoCl₂.6H₂O

P, B, Mo 100× Stock:

18.5 g KH₇PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O

Fe EDTA 100× Stock:

3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O

2,4-D Stock:

10 mg/mL Vitamin

B5 vitamins, 1000× Stock:

100.0 g mvo-inositol, 1.0 g nicotinic acid, 1.0 g pyridoxine HCl, 10 gthiamine.HCL.

Media (per Liter): SB199 Solid Medium:

1 package MS salts (Gibco/BRL—Cat. No. 11117-066), 1 mL B5 vitamins1000× stock, 30 g Sucrose, 4 ml 2, 4-D (40 mg/L final concentration), pH7.0, 2 gm Gelrite

SB1 Solid Medium:

1 package MS salts (Gibco/BRL—Cat. No. 11117-066), 1 mL B5 vitamins1000× stock, 31.5 g Glucose, 2 mL, 2, 4-D (20 mg/L final concentration),pH 5.7, 8 g TC agar

SB196:

10 mL of each of the above stock solutions 1-4, 1 mL B5 Vitamin stock,0.463 g (NH4)2 SO4, 2.83 g KNO3, 1 mL 2.4 D stock, 1 g asparagine, 10 gSucrose, pH 5.7

SB71-4:

Gamborg's B5 salts, 20 sucrose, 5 g TC agar, pH 5.7.

SB103:

1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock, 750 mgMgCl2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.

SB166:

SB103 supplemented with 5 g per liter activated charcoal.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting are picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds are sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles ofsterile distilled water and those less than 3 mm are placed onindividual microscope slides. The small end of the seed is cut and thecotyledons pressed out of the seed coat. Cotyledons are transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates are wrapped with fibertape. After this time, secondary embryos are cut and placed into SB196liquid media for 7 days.

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 50mL liquid medium SB196 on a rotary shaker, 150 rpm, 26° C. with coolwhite fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to twoweeks by inoculating approximately 35 mg of tissue into 50 mL of freshliquid SB196 (the preferred subculture interval is every 7 days).

Preparation of DNA for Bombardment:

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 pL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids are co-precipitated onto gold particles asfollows. The DNAs in suspension are added to 50 μL of a 10-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture is vortexed for 5 sec, spun ina microfuge for 5 sec, and the supernatant removed. The DNA-coatedparticles are then washed once with 150 μL of 100% ethanol, vortexed andspun in a microfuge again, then resuspended in 85 μL of anhydrousethanol. Five μL of the DNA-coated gold particles are then loaded oneach macrocarrier disk.

Tissue Preparation and Bombardment with DNA:

Approximately 150 to 250 mg of two-week-old suspension culture is placedin an empty 60 mm×15 mm petri plate and the residual liquid removed fromthe tissue using a pipette. The tissue is placed about 3.5 inches awayfrom the retaining screen and each plate of tissue is bombarded once.Membrane rupture pressure is set at 650 psi and the chamber is evacuatedto −28 inches of Hg. Following bombardment, the tissue from each plateis divided between two flasks, placed back into liquid media, andcultured as described above.

Selection of Transformed Embryos and Plant Regeneration:

After bombardment, tissue from each bombarded plate is divided andplaced into two flasks of SB196 liquid culture maintenance medium perplate of bombarded tissue. Seven days post bombardment, the liquidmedium in each flask is replaced with fresh SB196 culture maintenancemedium supplemented with 100 ng/mL selective agent (selection medium).For selection of transformed soybean cells the selective agent used canbe a sulfonylurea (SU) compound with the chemical name,2-chloro-N-((4-methoxy-6 methy-1,3,5-triazine-2-yl)aminocarbonyl)benzenesulfonamide (common names: DPX-W4189 and chlorsulfuron).Chlorsulfuron is the active ingredient in the DuPont sulfonylureaherbicide, GLEAN®. The selection medium containing SU is replaced everytwo weeks for 6-8 weeks. After the 6-8 week selection period, islands ofgreen, transformed tissue are observed growing from untransformed,necrotic embryogenic clusters. These putative transgenic events areisolated and kept in SB196 liquid medium with SU at 100 ng/mL foranother 2-6 weeks with media changes every 1-2 weeks to generate new,clonally propagated, transformed embryogenic suspension cultures.Embryos spend a total of around 8-12 weeks in contact with SU.Suspension cultures are subcultured and maintained as clusters ofimmature embryos and also regenerated into whole plants by maturationand germination of individual somatic embryos.

Somatic embryos became suitable for germination after four weeks onmaturation medium (1 week on SB166 followed by 3 weeks on SB103). Theyare then removed from the maturation medium and dried in empty petridishes for up to seven days. The dried embryos are then planted inSB71-4 medium where they are allowed to germinate under the same lightand temperature conditions as described above. Germinated embryos aretransferred to potting medium and grown to maturity for seed production.

Oil Analysis:

For oil analysis of somatic embryos, embryos were harvested aftertwo-three weeks of culture in the liquid maturation medium SB228 (SHaM).Approximately 30 events were created in transformations with pKR268,RTW218, RTW220, RTW221 and RTW222. All embryos generated for a givenevent were harvested in bulk and processed as follows. Embryos werefrozen on dry ice or by incubation in a −80° C. freezer for two hfollowed by lyophilization for 48 h.

Dried embryos were ground to a fine powder using a genogrinder vial(½″×2″ polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min. Groundembryo tissues were analyzed for their oil content by NMR method whichwas described as in US patent application 2008/0295204 A.

Embryo oil content was calculated as follows:

${\% \mspace{14mu} {oil}\mspace{14mu} \left( {\% \mspace{14mu} {wt}\mspace{14mu} {basis}} \right)} = {\frac{\left. {\left( {{NMR}\mspace{14mu} {{signal}/{sample}}\mspace{14mu} {wt}\mspace{14mu} (g)} \right) - 70.58} \right)}{351.45} \times 1.212056}$

For fatty acid composition analysis, aliquots of 30 to 50 mg of finepowdered somatic embryo tissues were weighed (to 0.0001 g precision)into 13×100 mm glass tubes fitted with Teflon® lined screw caps. Heptane(2 mL) was added to the powders in the tubes and after vortex mixingthey were placed into an ultrasonic bath (MR Scientific Model 750D)filled with water heated to 60° C. The samples were sonicated atfull-power (˜360 W) for 15 min and were then centrifuged (5 min×1700 g),The supernatants were transferred to clean 13×100 mm glass tubes and thepellets were extracted 2 more times with heptane (2 mL, secondextraction, 1 mL, third extraction). The supernatants from the threeextractions were combined. Five hundred micro liters of the pooledextracts were transferred to clean 13×100 mm glass tubes. Internalstandard [10 μL; 10 mg triheptadecanoin (Nu-Chek Prep, Elysian, Minn.,USA)/mL toluene] was added to each tube followed by 1 mL 1% sodiummethoxide (v/v in anhydrous methanol). The tubes were capped withTeflon® lined closures and after thorough vortex mixing the samples wereheated at 50° C. for 30 min. After cooling to room temperature 1 mL, 25%(wt/v) sodium chloride and 1 mL of heptane were added to each sample.The tubes were vortex mixed and after centrifugation (3 min at 1700×g) aportion of the upper organic phase was transferred to a sample vial forGC analysis. Analysis was performed on an Agilent 6890 gas chromatographfitted with an Omegawax 320 capillary column (30 m, 0.32 mm OD, 0.25 μmfilm thickness; Supelco, Bellefonte Pa., USA). Detection was by FlameIonization. Relative peak areas were used to calculate the fatty acidprofiles and quantitation was performed relative to the internalstandard. Results of these experiments are summarized in FIG. 12 andTable 6.

TABLE 6 Oil increase by sucrose transporters co-expressed with YLDGAT1(5 events each from lowest-oil-accumulating events and highest-oil-accumulating events or %18:1 events were averaged and the difference wascalculated). %18:1 levels were calculated in a similar manner. Avg AvgDiffer- Differ- Low High ence Avg Avg ence % % in % Low High in Oil OilOil %18:1 %18:1 %18:1 pKR268 4.4 9.6 5.2 16.59 21.89 5.30 (vector only)RTW218 6.3 13.5 7.2 24.82 36.38 11.56 (YLDGAT 1) RTW220 7.1 13.7 6.620.58 34.43 13.84 (YLDGAT1 and ATSUC2) RTW221 5.5 15.4 9.9 17.71 34.5316.82 (YLDGAT1 and ATSUT4) RTW222 7.5 15.6 8.1 20.5 36.57 16.07 (YLDGAT1and ATSUT2)

In summary, as disclosed previously, YLDGAT1 gene expressionconsistently increased total oil level as well as oleic acid content insoybean somatic embryos when compared to the control transgenic lineswhich carried only the native vector construct. Co-expression of highaffinity, SUT1-type transporter, ATSUC2 and YLDGAT1 gene yielded similarlevels of oil and oleic acid to events expressing the YLDGAT1 genealone. However, co-expression of low to medium affinity SUT2 andSUT4-type transporters, ATSUT2 and ATSUT4 showed significant additiveeffect by further increasing oil and oleic levels compared to transgenicembryos with only YLDGAT1 gene. Taken together these findings stronglysuggest that co-expression of high capacity sucrose transportersincluding ATSUT2 and ATSUT4 with YLDGAT genes provides an efficientstrategy to achieve an increase in the total oil content of soybeanseed.

Example 8 Co-Expression of Yarrowia lipolytica DGAT Genes and LowAffinity, High Capacity Sucrose Transporter Genes in Soybean Seed

A DNA construct for co-expression of YLDGAT1 and a hexa-histidine taggedversion of Arabidopsis SUT4 (At1g09960) was generated as follows: Aplasmid, RTW212 (SEQ ID NO: 138), containing a soybean selection markercassette which was composed of soybean SAMS promoter, soybean HRA gene,and soybean ALS terminator, was used to construct RTW227 vector. ATSUT4expression cassette was derived from RTW162p1 (ann sut4) (SEQ ID NO:135) described in Example 7, RTW162p1 (ann sut4) was digested with SbfIand a fragment containing soybean annexin promoter, ATSUT4 gene, andsoybean myb2 terminator, was gel-purified. The fragment was ligated toSbfI linearized, dephosphorylated RTW212 plasmid to give RTW226 (SEQ IDNO: 139). RTW218, which was described in Example 7, was a source forYL-DGAT1 expression cassette containing soybean GY1 promoter, YL-DGAT1gene, and phaseolin terminator. RTW218 was digested with BsiWI and SaltSall site was completely filled using Klenow polymerase reaction. Afragment containing YL-DGAT1 expression cassette was then gel-purifiedand ligated into SmaI/BsiWI digested, dephosphorylated RTW226. Theresultant plasmid was set forth as RTW227F (ATSUT4 DGAT1 ALS), PHP36710(SEQ ID NO: 140).

Soybeans were transformed as shown in Example 7.

Somatic embryos became suitable for germination after four weeks andwere then removed from the maturation medium and dried in empty petridishes for one to five days. The dried embryos were then planted inSB71-4 medium where they were allowed to germinate under the same lightand temperature conditions as described above. Germinated embryos weretransferred to sterile soil and grown to maturity for seed production.

A total of 69 T₀ plants derived from 42 transgenic events were generatedwith a 10.5 kb AscI restriction fragment of PHP36710 at concentration of15 pg per bp of plasmid DNA per gold particle preparation (see above).For every T₀ plant 8 seed were initially screened for the absence orpresence of the transgene-derived YLDGAT by assaying the seed fatty acidcomposition. Seed oil content was measured by NMR as described in USpatent application 2008/0295204 A. US patent application 2008/0295204 A1also discloses that expression of Yarrowia DGAT genes in transgenicsoybean somatic embryos and soybean seed was associated with increasedincorporation of oleic acid into the total esterified fatty acidfraction which on the other hand was tightly correlated with totalaccumulation of fatty acids is this tissue. In T1 seed derived from 27of the total 42 events generated with PHP36710 the R² value expressingcorrelation between oleic acid and total fatty acid content was >0.2. Inthese events the R² related to the correlation between oleic acidcontent and total fatty acid content ranged from 0.21 to 0.98. T1 seedfrom four events were analyzed in more detail. Results are shown inTable 7 where n is the number of seeds analyzed, avg % oil <18% oleic isthe average oil content of seed weight) of all seeds with an oleic acidcontent of less than 18% of the total fatty acid content, avg % oil >18%oleic is the average oil content (% of seed weight) of all seeds with anoleic acid content that is equal to or greater than 18% of the totalfatty acid content, delta % points is the difference in oil content (%points) between seeds with an oleic acid content that is equal to orgreater than 18% of the total fatty acid content and seeds with an oleicacid content of less than 18% of the total fatty acid content, delta %is the difference in oil content (%) of seed with an oleic acid contentthat is equal to or greater than 18% of the total fatty acid content andseeds with an oleic acid content of less than 18% of the total fattyacid content, and R2% oleic/% oil is the correlation coefficient for therelationship between oleic acid content (% of total fatty acids) and oilcontent (% of seed) for T1 seed of a given transgenic event.

TABLE 7 Oil Content of T1 Soybean Seed Generated with PHP 36710 avg %avg % oil < oil ≥ delta R² % 18% 18% % delta oleic/% n oleic n oleicpoints % oil AFS 5925.1.9.2 5 17.2 19 22.1 4.9 28.6 0.71 AFS 5925.2.7.17 18.6 17 23.4 4.8 25.5 0.28 AFS 5925.1.6.1 5 19.1 19 22.0 2.9 15.1 0.57AFS 5925.1.6.1 7 20.6 17 23.1 2.5 12.2 0.61

In summary applicants have demonstrated that co-expression of DG.ATgenes and SUT2 or SUT4 sucrose transporter genes provides an efficientmethod to increase the total fatty acid content of seed.

Example 9 Cloning Soybean Sucrose Transporter Genes for Co-Expressionwith DGAT Genes in Soy

The present example describes cloning of soy Sut2 and Sut4 homologs forco-expression with DGAT genes.

One skilled in the art will appreciate that a number of molecularbiology techniques exist for cloning soy genes from cDNA or cDNAlibraries. Most techniques involve isolating total RNA from soy tissuefollowed by either direct synthesis of cDNA from total RNA orpurification of mRNA first followed by cDNA synthesis, For example,total RNA can be isolated from soy tissue using TRIzol® Reagent(Invitrogen Corporation, Carlsbad, Calif.) and following themanufacturer's protocol provided. mRNA can be isolated from total RNAusing the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.)following the manufacturer's protocol provided, A cDNA library can begenerated from total RNA or mRNA using the Cloneminer™ cDNA LibraryConstruction Kit (Cat. No.18249-029, Invitrogen Corporation, Carlsbad,Calif.) and following the manufacturer's protocol provided (Version B,25-0608). cDNA libraries may also be prepared using Uni-ZAP™ XR vectorsaccording to the manufacturer's protocol (Stratagene Cloning Systems, LaJolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts are contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut Bluescript II SK(±) vectors (Stratagene) using T4 DNA ligase (NewEngland Biolabs), followed by transfection into DH10B cells according tothe manufacturer's protocol (GIBCO BRL Products). Normalized cDNAlibraries can also be prepared, for example as described in U.S. Pat.No. 5,482,845, incorporated herein by reference.

Glyma08g40980 (SEQ ID NO: 33) is amplified from soy cDNA or a soy cDNAlibrary using oligonucleotides GmSut2-1For (SEQ ID NO: 141) andGmSut2-1Rev (SEQ ID NO: 142), designed to introduce NotI site flankingthe gene, along with a suitable DNA polymersase such as GoTaq polymerase(Promega, USA) or Phusion polymerase (New England Biolabs, Inc.,Ipswich, Mass.) and following the manufacturer's protocol. The resultingPCR product is cloned into a suitable cloning vector such as thepCR-Blunt® cloning vector using the Zero Blunt® PCR Cloning Kit(Invitrogen Corporation), following the manufacturer's protocol whenPhusion polymerase is used or into pGEM®-T Easy Vector (Promega),following the manufacturer's protocol, when GoTaq polymerase is used.

In a similar way, Glyma18g15950 (SEQ ID NO: 35) is amplified witholigonucleotides GmSut2-2For (SEQ ID NO: 143) and GmSut2-2Rev (SEQ IDNO: 144), Glyma02g38300 (SEQ ID NO: 37) is amplified witholigonucleotides SA150 (SEQ ID NO: 145) and SA151 (SEQ ID NO: 146) andGlyma04g09460 (SEQ ID NO: 39) is amplified with oligonucleotides SA148(SEQ ID NO: 147) and SA149 (SEQ ID NO: 148).

Alternatively, soy Sut genes flanked by Not I restriction sites can besynthesized directly by companies such as, but not limited to, DNA 2.0(California, USA), Codon Devices (MA, USA) or by GENEART AG (Regensburg,Germany).

In this way, soy Sut homolog genes flanked by NotI sites can be easilycloned into suitable soy expression vectors.

For example, NotI fragment of a soy Sut homolog gene can be ligated intoa vector, which has an expression cassette composed of annexin promoterand soybean myb2 terminator. Such vector can be RTW162p1. as describedin Example 7. Then the cassette can be transferred to any plasmids thatcan be used either for soybean somatic embryonic transformation orsoybean stable transfoi

Example 10 Co-Expression of Sucrose Transporters and Yarrowia lipolyticaDGAT Genes in Maize

Co-expression of sucrose transporters such as those listed in, but notlimited to, Table 4 or SUT2, or SUT4 homologs in Table 3 with DGAT genescan be used in the seeds of maize to increase the oil content of thistissue. As described below, this result can be achieved by transformingmaize with expression cassettes comprising an open reading frame of DGATgenes operably linked on their 5′ ends to embryo preferred promoters,such as the promoter for the maize 16 kDa oleosin gene (Lee, K. andHuang, A. H., Plant Mol. Biol. 26:1981-1987 (1984)) and maize embryoabundant (EAP1) promoter and terminator (US 2006272058 A1), andsimilarly configured sucrose transporter genes.

For example, an expression cassette comprising the promoter from themaize 16 kDa oleosin gene (OLE PRO), the coding sequence of the YLDGATgene and the polyadenylation signal sequence/terminator from thenopaline synthase (NOS) gene of Agrobacterium tumefaciens is constructedusing methods and technologies known in the art. A second expressioncassette comprises a sucrose transporter gene under the transcriptionalcontrol of the maize embryo abundant protein (EAP1) promoter andterminator, with the maize ADH1 INTRON1 inserted between the promoterand coding sequence for enhanced expression. The two expressioncassettes are linked, together with a gene encoding a selectable marker,in a binary vector suitable for Agrobacterium-mediated transformation ofmaize.

An Agrobacterium-based protocol can be used for the transformation ofmaize (see below). The resulting binary vector is introduced intoAgrobacterium LBA4404 (PHP10523) cells, preferably by electroporation.An in vivo recombination generates a cointegrate plasmid between theintroduced binary vector and the vir plasmid (PHP10523) resident in theAgrobacterium cells. The resulting Agrobacterium cells are used totransform maize.

Transformation of Maize Mediated by Agrobacterium:

Freshly isolated immature embryos of maize, about ten days afterpollination (DAP), can be incubated with the Agrobacterium. Thepreferred genotype for transformation is the highly transformablegenotype Hi-II (Armstrong, Maize Gen. Coop. Newsletter 65:92-93 (1991)).An F1 hybrid created by crossing a Hi-II with an elite inbred may alsobe used. After Agrobacterium treatment of immature embryos, the embryoscan be cultured on medium containing toxic levels of herbicide. Onlythose cells that receive the herbicide resistance gene, and the linkedgene(s), grow on selective medium. Transgenic events so selected can bepropagated and regenerated to whole plants, produce seed, and transmittransgenes to progeny.

Preparation of Agrobacterium:

The engineered Agrobacterium tumefaciens LBA4404 can be constructed tocontain plasmids for seed-preferred expression of DGAT and sucrosetransporter genes, as disclosed in U.S. Pat. No. 5,591,616 (the contentsof which are hereby incorporated by reference). To use the engineeredconstruct in plant transformation, a master plate of a single bacterialcolony transformed with plasmids for seed-preferred expression of bothgenes can be prepared by inoculating the bacteria on minimal AB mediumand allowing incubation at 28° C. for approximately three days. (Thecomposition and preparation of minimal AB medium has been previouslydescribed in PCT Publication No. WO 02/009040 (the contents of which arehereby incorporated by reference). A working plate can then be preparedby streaking the transformed Agrobacterium on YP medium (0.5% (w/v)yeast extract, 1% (w/v) peptone, 0.5% (w/v) sodium chloride, 1.5% (w/v)agar) that contains 50 μg/mL of spectinomycin.

The transformed Agrobacterium for plant transfection and co-cultivationcan then be prepared one day prior to maize transformation. Into 30 mLof minimal A medium (prepared as described in PCT Publication Nb. WO02/009040) in a flask was placed 50 μg/mL spectinomycin, 100 μMacetosyringone, and about a ⅛ loopful of Agrobacterium from a one totwo-day-old working plate. The Agrobacterium can then be grown at 28° C.with shaking at 200 rpm for approximately fourteen h. At mid-log phase,the Agrobacterium can be harvested and resuspended at a density of 3 to5×108 CFU/mL in 561Q medium that contains 100 μM acetosyringone usingstandard microbial techniques. The composition and preparation of 561Qmedium was described in PCT Publication No. WO 02/009040.

Immature Embryo Preparation:

Nine to ten days after controlled pollination of a maize plant,developing immature embryos are opaque and 1-1.5 mm long. This length isthe optimal size for infection with the Agrobacterium. The husked earscan be sterilized in 50% commercial bleach and one drop Tween-20 forthirty minutes, and then rinsed twice with sterile water. The immatureembryos can then be aseptically removed from the caryopsis and placedinto 2 mL of sterile holding solution consisting of medium 561Q thatcontains 100 μM of acetosyringone.

Agrobacterium Infection and Co-Cultivation of Embryos:

The holding solution can be decanted from the excised immature embryosand replaced with transformed Agrobacterium. Following gentle mixing andincubation for about five minutes, the Agrobacterium can be decantedfrom the immature embryos. Immature embryos were then moved to a plateof 562P medium, the composition of which has been previously describedin PCT Publication No. WO 02/009040. The immature embryos can be placedon this media scutellum surface pointed upwards and then incubated at20° C. for three days in darkness. This step can be followed byincubation at 28° C. for three days in darkness on medium 562P thatcontains 100 μg/mL carbenecillin as described in U.S. Pat. No.5,981,840.

Selection of Transgenic Events:

Following incubation, the immature embryos can be transferred to 5630medium, which can be prepared as described in PCT Publication No. WO02/009040. This medium contains Bialaphos for selection of transgenicplant cells as conferred by the BAR gene that is linked to barley HGGTexpression cassette. At ten to fourteen-day intervals, embryos weretransferred to 5630 medium. Actively growing putative transgenicembryogenic tissue can be after six to eight weeks of incubation on the5630 medium.

Regeneration of T₀ Plants:

Transgenic embryogenic tissue is transferred to 288W medium andincubated at 28° C. in darkness until somatic embryos matured, or aboutten to eighteen days. Individual matured somatic embryos withwell-defined scutellum and coleoptile are transferred to 272 embryogermination medium and incubated at 28° C. in the light. After shootsand roots emerge_(;) individual plants are potted in soil andhardened-off using typical horticultural methods.

288W medium contains the following ingredients: 950 mL, of deionizedwater; 4.3 g of MS Salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSVitamins Stock Solution (Gibco); 1 mL of zeatin (5 mg/mL solution); 60 gsucrose; 8 g of agar (Sigma A-7049, Purified), 2 mL of indole aceticacid (0.5 mg/mL solution*); 1 mL of 0.1 mM ABA*; 3 mL of Bialaphos (1mg/mL solution*); and 2 mL of carbenicillin (50 mg/mL solution). The pHof this solution is adjusted to pH 5.6. The solution is autoclaved andingredients marked with an asterisk (*) are added after the media hascooled to 60° C.

Medium 272 contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSvitamins stock solution (Gibco); 40 g of Sucrose; and 1.5 g of Gelrite.This solution is adjusted to pH 5.6 and then autoclaved.

Example 11 Analysis of Kernel Oil Content Nuclear Magnetic Resonance(NMR) Analysis

Seed are imbibed in distilled water for 12-24 hours at 4° C. The embryois dissected away and stored in a 48 well plate. The samples arelyophilized over-night in a Virtis 24×48 lyophilizer. The NMR (ProcessControl Technologies—PCT (Ft. Collins, Colo.)) is set up as per themanufacturer's instructions. The NMR is calibrated using a series of 5mm NMR tubes containing precisely measured amounts of corn oil (Mazola).The calibration standards are 3, 6, 9, 12, 15, 18, 21, 27, 33, and 40 mgof oil.

Example 12 Introduction of SUT2 and SUT4 Sucrose Transporters into aHigh Oil Plant

SUT2 or SUT4 sucrose transporters can be introduced into a high oilplant by crossing a first plant with a naturally occurring high oilphenotype explained in Example 1 to a second plant with alteredexpression of SUT2 or SUT 4 explained in Example 1. The progenyresulting from the cross can be evaluated for a further increase in oilproduction and overexpression of SUT2 or SUT 4 sucrose transporters canbe compared to said first plant.

Furthermore, sucrose transporters can be introduced into a high oilplant cell by introduction of a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory element,wherein said polynucleotide encodes a polypeptide that is a SUT2 or SUT4 sucrose transporter. A transgenic plant can be regenerated from theregenerable plant cell wherein the transgenic plant comprises in itsgenome the recombinant DNA construct. A progeny plant can be derivedfrom the transgenic plant and can be selected for expression of SUT2 orSUT 4 sucrose transporters.

Example 13 Co-Expression of Sucrose Transporters and Yarrowia lipolyticaDGAT Genes in Oilseed Plants

Overexpression of exogenous or endogenous sucrose transporter genes incombination with Yarrowia lipolytica DGAT genes can be performed in anyoilseed plant.

Example 14 Co-Expression of Sucrose Transporters and Yarrowia lipolyticaDGAT1 in Soy

The present example describes cloning soy Sut4 and Sut2 genes into soyexpression vectors and co-expressing with the Yarrowia lipolytica DGAT1gene (YLDGAT1) in soy somatic embryos. In all cases, each Sut gene isunder control of the soy annexin promoter and YLDGAT1 is under controlof the soy glycinin Gy1 promoter as described for expression vectors inExample 7.

Construction of pKR1684 (GmSut4-1 YLDGAT1 35Hyg)

Glyma02g38300 (SEQ ID NO:37), also called GmSut4-1, was PCR amplifiedfrom a soy cDNA library using oligonucleotides SA150 (SEQ ID NO: 145)and SA151 (SEQ ID NO: 146) and GoTaq polymerase as described in Example9. The resulting DNA fragment was cloned into pGEM®-T Easy Vector(Promega), following the manufacturer's protocol, to produceGlyma02g38300 in pGEM-T Easy (SEQ ID NO: 151).

The NotI fragment of Glyma02g38300 in pGEM-T Easy (SEQ ID NO: 151),containing GmSut4-1, was cloned into the NotI site of RTW147 (SEQ ID NO:132) to produce pKR1680 (SEQ ID NO: 152).

The PstI fragment of pKR1680 (SEQ ID NO: 152), containing GmSut4-1, wascloned into the SbfI site of RTW218 (SEQ ID NO: 128) to produce pKR1684(SEQ ID NO: 153).

Construction of pKR1685 (GmSut2-1 YLDGAT1 35Hyg)

Glyma08g40980 (SEQ ID NO: 33), also called GmSut2-1, was PCR amplifiedfrom a soy cDNA library using oligonucleotides GmSut2-1For (SEQ ID NO:141) and GmSut2-1Rev (SEQ ID NO: 142) and Phusion polymerase asdescribed in Example 9. The resulting DNA fragment was cloned into ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pLF235 (SEQ ID NO: 154).

The NotI fragment of pLF235 (SEQ II) NO: 154), containing GmSut2-1, wascloned into the NotI site of RTW147 (SEQ ID NO: 132) to produce pKR1681(SEQ ID NO: 155).

The PstI fragment of pKR1681 (SEQ ID NO: 155), containing GmSut2-1, wascloned into the SbfI site of RTW218 (SEQ ID NO: 128) to produce pKR1685(SEQ ID NO: 156).

Construction of pKR1686 (GmSut4-2 YLDGAT1 35Hyg)

Glyma04g09460 (SEQ ID NO: 39), also called GmSut4-2, was PCR amplifiedfrom a soy cDNA library using oligonucleotides GmSut4-2For (SEQ ID NO:157) and GmSut4-2Rev (SEQ ID NO: 158) and Phusion polymerase asdescribed in Example 9. The resulting DNA fragment was cloned into ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce pLF236 (SEQ ID NO: 159).

The NotI fragment of pLF236 (SEQ ID NO: 159), containing GmSut4-2, wascloned into the NotI site of RTW147 (SEQ H) NO: 132) to produce pKR1682(SEQ ID NO: 160),

The SbfI fragment of pKR1682 (SEQ ID NO: 160), containing GmSut4-2, wascloned into the SbfI site of RTW218 (SEQ ID NO: 128) to produce pKR1686(SEQ ID NO: 161).

Soybean embryonic suspension cultures (cv. Jack) were initiated andmaintained as described in Example 7. Soybean embryonic suspensioncultures were transformed with RTW218 (SEQ ID NO: 128, Example 7),pKR1684, pKR1685 or pKR1686, by particle gun bombardment as described inExample 7 with the following modifications.

Preparation of DNA for Bombardment:

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution (either intactplasmid or DNA fragment prepared as described above), 50 μL 2.5M CaCl₂and 20 μL of 0.1 M spermidine. The mixture was pulsed 5 times on level 4of a vortex shaker and spun for 5 sec in a bench microfuge. After a washwith 150 μL of 100% ethanol, the pellet was suspended by sonication in85 μL of 100% ethanol, Five μL of DNA suspension was dispensed to eachflying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μLaliquot contained approximately 0.058 mg gold particles per bombardment(i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25 mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker. Specifically, following bombardment, the tissue was placed intofresh SB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB196 is exchanged with fresh SB196 containing 30mg/L hygromycin. The selection media was refreshed weekly. Four to sixweeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were matured in soybeanhistodifferentiation and maturation liquid medium (SHaM liquid media;Schmidt et al., Cell Biology and Morphogenesis 24:393 (2005)), using amodified procedure. Briefly, after 4 weeks of selection in SB196, asdescribed above, embiyo clusters are removed to 35 mL of SB228 (SHaMliquid media) in a 250 mL Erlenmeyer flask. Tissue is maintained in SHaMliquid media on a rotary shaker at 130 rpm and 26° C., with cool whitefluorescent lights on a 16:8 hr day/night photoperiod at a lightintensity of 60-85 μE/m2/s for 2 weeks as embryos matured. Embryos grownfor 2 weeks in SHaM liquid media are equivalent in size and fatty acidcontent to embryos cultured on SB166/SB103 for 5-8 weeks.

SHaM Media Recipes:

SB 228- Soybean Histodifferentiation & Maturation (SHaM) (per liter) DDIH2O 600 mL FN-Lite Macro Salts for SHaM 10X 100 mL MS Micro Salts 1000x1 mL MS FeEDTA 100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mLL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mLpH 5.8 Autoclave Add to cooled media (≤30° C.): *Glutamine (Final conc.30 mM) 4% 110 mL *Note: Final volume will be 1010 mL after glutamineaddition.Because glutamine degrades relatively rapidly, it may be preferable toadd immediately prior to using media. Expiration 2 weeks after glutamineis added; base media can be kept longer w/o glutamine.

FN-lite Macro for SHAM 10X- Stock #1 (per liter) (NH₄)2SO₄ (AmmoniumSulfate) 4.63 g KNO₃ (Potassium Nitrate) 28.3 g MgSO₄*7H₂0 (MagnesiumSulfate Heptahydrate)  3.7 g KH₂PO₄ (Potassium Phosphate, Monobasic)1.85 g Bring to volume Autoclave

MS Micro 1000X- Stock #2 (per 1 liter) H₃BO₃ (Boric Acid) 6.2 gMnSO₄*H₂O (Manganese Sulfate Monohydrate) 16.9 g ZnSO4*7H20 (ZincSulfate Heptahydrate) 8.6 g Na₂MoO₄*2H20 (Sodium Molybdate Dihydrate)0.25 g CuSO₄*5H₂0 (Copper Sulfate Pentahydrate) 0.025 g CoCl₂*6H₂0(Cobalt Chloride Hexahydrate) 0.025 g KI (Potassium Iodide) 0.8300 gBring to volume Autoclave

FeEDTA 100X- Stock #3 (per liter) Na₂EDTA* (Sodium EDTA) 3.73 gFeSO₄*7H₂0 (Iron Sulfate Heptahydrate) 2.78 g *EDTA must be completelydissolved before adding iron. Bring to Volume Solution isphotosensitive. Bottle(s) should be wrapped in foil to omit light.Autoclave

Ca 100X- Stock #4 (per liter) CaCl₂*2H₂0 (Calcium Chloride Dihydrate) 44g Bring to Volume Autoclave

B5 Vitamin 1000X- Stock #5 (per liter) Thiamine*HCl 10 g Nicotinic Acid1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store frozen

4% Glutamine- Stock #6 (per liter) DDI water heated to 30° C. 900 mLL-Glutamine 40 g Gradually add while stirring and applying low heat. Donot exceed 35° C. Bring to Volume Filter Sterilize Store frozen *

After maturation in SHaM liquid media, approximately 10 embyros perevent are frozen at −80° C., lyophilized and analyzed for oil contentand fatty acid profile as described in Example 7.

Results showing oil content and fatty acid profile for approximately 30transgenic soybean lines (events) from each experiment transformed withRTW218, pKR1684, pKR1685 or pKR1686 are shown in Tables 8, 9, 10 or 11,respectively. Average oil content and fatty acid profile for all eventsin an experiment is shown in each table as Avg. Average oil content andfatty acid profile for 5 events having highest oil content in anexperiment is shown in each Table as Avg-Top5. In each table, events aresorted based on decreasing oil conte

TABLE 8 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with RTW218 (YLDGAT1 only). MSE2692 (RTW218) - YLDGAT1 EventOil 16:0 18:0 18:1 18:2 18:3 2692-2 10.5 12.9 6.0 32.9 39.0 9.2 2692-1510.1 14.0 5.9 29.5 41.0 9.7 2692-12 9.9 13.2 5.9 28.9 41.4 10.6 2692-89.4 13.8 7.8 31.9 36.7 9.7 2692-16 9.2 11.5 6.4 35.3 35.0 11.7 2692-218.7 13.7 7.6 32.3 36.9 9.5 2692-27 8.3 13.6 7.3 32.5 35.1 11.5 2692-308.2 13.4 6.0 30.0 38.4 12.2 2692-29 8.0 13.4 7.2 30.8 38.1 10.5 2692-187.9 14.6 6.3 25.7 41.8 11.6 2692-25 7.6 14.0 8.1 31.0 35.5 11.4 2692-207.4 14.5 5.8 24.9 40.8 13.9 2692-19 7.4 14.7 6.2 27.7 39.0 12.4 2692-37.2 14.6 6.6 26.5 40.2 12.2 2692-28 6.9 16.3 6.7 23.2 40.9 12.9 2692-96.7 15.2 7.0 24.2 40.1 13.4 2692-17 6.3 15.3 6.8 23.9 40.5 13.5 2692-46.1 16.4 5.6 17.8 44.3 15.9 2692-10 5.5 15.8 6.1 24.6 39.6 13.8 2692-145.3 16.1 6.6 25.0 38.4 13.8 2692-23 5.0 17.2 5.3 16.8 42.6 18.1 2692-15.0 16.6 5.8 15.2 44.2 18.2 2692-13 4.3 15.9 5.6 21.8 39.9 16.8 2692-54.1 16.0 6.2 20.2 40.6 17.0 2692-11 3.8 17.9 6.8 19.6 39.5 16.2 2692-73.7 16.1 5.6 20.1 39.2 18.9 2692-31 3.5 17.4 5.3 19.2 39.4 18.7 2692-63.2 16.3 5.6 15.8 41.3 21.0 2692-26 3.2 18.4 7.6 19.6 36.6 17.7 2692-243.0 17.1 5.0 15.5 39.7 22.6 2692-22 1.7 15.4 6.0 16.1 41.1 21.4 Avg. 6.415.2 6.4 24.5 39.6 14.4 Avg.-Top5 9.8 13.1 6.4 31.7 38.6 10.2

TABLE 9 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1684 (YLDGAT1 & GmSut4-1). MSE2689 (pKR1684) -YLDGAT1 & GmSut4-1 Event Oil 16:0 18:0 18:1 18:2 18:3 2689-8 10.8 15.17.7 34.6 34.9 7.8 2689-5 10.5 13.0 6.4 34.5 35.3 10.8 2689-14 10.3 14.06.7 33.4 35.6 10.3 2689-11 10.2 13.2 7.0 35.5 33.5 10.8 2689-22 9.9 13.36.2 31.2 38.2 11.1 2689-19 9.3 14.0 6.5 35.0 34.6 9.9 2689-13 9.3 14.35.9 27.1 40.6 12.1 2689-4 8.5 12.8 5.9 30.7 40.0 10.6 2689-23 8.5 14.56.7 29.6 37.1 12.1 2689-1 8.4 13.2 7.0 35.3 34.6 9.9 2689-24 8.3 13.37.5 34.8 33.3 11.1 2689-9 8.1 13.6 6.7 33.5 35.5 10.7 2689-17 8.0 13.76.7 29.1 38.1 12.4 2689-28 7.8 14.1 6.6 29.8 37.4 12.1 2689-10 7.8 14.06.4 29.2 37.6 12.8 2689-29 7.5 14.2 6.4 26.4 41.5 11.4 2689-3 7.4 14.85.8 18.5 46.0 14.8 2689-26 6.4 14.7 6.5 26.7 37.7 14.5 2689-2 6.3 14.36.8 31.8 35.6 11.5 2689-21 6.0 16.2 5.0 16.9 44.9 17.0 2689-6 5.8 14.67.0 29.4 35.9 13.2 2689-12 5.5 16.3 6.8 19.7 40.7 16.5 2689-25 5.4 16.25.9 18.0 41.8 18.0 2689-20 5.2 15.4 5.5 23.9 38.5 16.8 2689-15 5.2 15.05.9 26.3 37.4 15.5 2689-16 5.0 16.3 6.6 19.6 38.1 19.4 2689-27 4.7 17.94.9 16.5 41.5 19.1 2689-7 4.3 16.7 6.2 22.9 36.1 18.1 2689-30 4.1 17.46.8 21.8 37.5 16.5 2689-18 3.8 15.5 5.8 19.1 39.4 20.2 Avg. 7.3 14.7 6.427.4 38.0 13.6 Avg.-Top5 10.3 13.7 6.8 33.8 35.5 10.2

TABLE 10 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1685 (YLDGAT1 & GmSut2-1). MSE2690 (pKR.1685) -YLDGAT1 & GmSut2-1 Event Oil 16:0 18:0 18:1 18:2 18:3 2690-9 13.1 11.95.9 34.7 39.4 8.1 2690-2 12.0 12.6 7.1 32.3 39.2 8.8 2690-28 11.6 14.34.8 29.6 41.3 10.0 2690-13 11.4 13.5 5.2 29.6 41.0 10.7 2690-30 11.313.1 7.4 34.7 35.2 9.7 2690-10 11.1 12.4 6.3 31.8 39.5 10.0 2690-1 10.913.5 6.1 29.0 41.0 10.4 2690-20 10.7 13.1 7.1 32.8 37.7 9.4 2690-18 10.712.3 8.2 31.1 38.1 10.4 2690-29 10.2 13.1 6.7 31.2 38.7 10.4 2690-1710.1 14.2 5.6 31.0 38.4 10.8 2690-7 9.8 13.4 5.4 30.4 39.0 11.8 2690-249.6 14.8 5.6 30.0 39.2 10.4 2690-6 9.6 13.9 6.7 33.0 35.1 11.4 2690-159.4 12.9 6.6 35.1 35.8 9.5 2690-31 9.2 14.9 5.4 25.0 42.0 12.7 2690-229.2 13.4 6.1 29.9 38.5 12.1 2690-21 9.1 14.0 6.5 28.6 39.0 11.9 2690-278.6 13.1 7.1 34.0 36.1 9.7 2690-5 8.4 14.6 5.6 26.3 40.9 12.6 2690-258.2 14.5 6.5 27.3 39.1 12.7 2690-23 7.8 15.0 6.5 25.1 40.3 13.0 2690-117.8 15.6 5.6 23.7 42.0 13.2 2690-12 7.3 15.3 5.0 25.2 40.4 14.1 2690-36.7 16.8 5.0 16.7 44.2 17.2 2690-16 5.5 15.0 7.0 19.0 42.5 16.5 2690-85.3 18.4 4.9 15.5 42.7 18.5 2690-14 4.9 16.0 5.0 16.4 43.3 19.3 2690-44.7 17.2 5.1 16.3 40.9 20.4 2690-19 4.6 16.2 6.4 19.2 41.0 17.3 2690-263.0 18.3 4.9 17.3 37.3 22.2 Avg. 8.8 14.4 6.0 27.2 39.6 12.7 Avg.-Top511.9 13.1 6.1 32.2 39.2 9.5

TABLE 11 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1686 (YLDGAT1 & GmSut4-2). MSE2691 (pKR1686) -YLDGAT1 & GmSut4-2 Event Oil 16:0 18:0 18:1 18:2 18:3 2691-19 12.9 12.67.5 36.9 34.8 8.3 2691-16 12.4 12.2 6.7 33.5 38.4 9.2 2691-23 12.0 12.87.5 37.6 33.6 8.5 2691-28 11.8 13.1 5.6 26.9 44.4 10.1 2691-11 11.6 13.66.3 27.5 42.0 10.6 2691-22 10.9 13.8 5.9 26.9 42.3 11.2 2691-31 10.813.3 6.6 27.7 42.4 10.1 2691-30 10.1 15.4 5.8 25.4 41.8 11.5 2691-1210.1 13.0 7.1 31.3 38.9 9.6 2691-25 9.9 15.6 6.9 23.7 42.4 11.4 2691-279.8 13.5 7.7 31.5 37.5 9.8 2691-10 9.6 12.5 7.3 31.2 39.1 9.9 2691-299.5 17.0 4.4 17.3 47.4 13.9 2691-18 9.1 14.4 7.0 30.8 36.9 10.8 2691-88.8 16.9 4.8 17.5 46.9 13.9 2691-4 8.1 17.4 4.7 18.5 44.5 14.9 2691-147.8 13.9 6.5 26.7 40.0 12.8 2691-15 7.7 15.1 6.9 27.0 38.0 13.1 2691-246.9 14.4 6.9 28.7 35.5 14.5 2691-9 6.8 14.9 6.1 27.2 38.4 13.4 2691-56.8 17.6 5.3 17.7 43.0 16.4 2691-1 6.7 16.6 5.5 20.1 43.5 14.3 2691-265.9 17.3 5.4 16.8 43.4 17.2 2691-21 5.8 17.7 5.7 18.4 42.0 16.1 2691-35.6 17.4 4.7 14.4 44.5 19.0 2691-17 5.3 16.3 6.4 21.6 38.5 17.1 2691-135.0 16.8 6.5 22.1 38.4 16.2 2691-20 4.9 15.6 5.7 17.3 43.3 18.1 2691-64.8 16.4 6.2 17.0 41.4 19.1 2691-7 4.0 15.9 5.5 16.3 41.2 21.1 2691-24.0 18.8 5.8 17.6 39.9 18.0 Avg. 8.2 15.2 6.2 24.3 40.8 13.5 Avg.-Top512.1 12.9 6.7 32.5 38.6 9.3

A summary of the average oil content and fatty acid profile for the 5events having highest oil content for each experiment is shown in Table12. Also shown is the change in oil content compared to the YLDGAT1 onlyexperiment (dOil) as well as the percent increase in oil compared to theYLDGATI only experiment (% dOil).

TABLE 12 Average Oil Content and Fatty Acid Profiles for 5 Events havinghighest oil content in each experiment. Average Oil Content and FattyAcid Profile for Top5 Events per Experiment MSE Vector Gene 1 Gene 2 OildOil % dOil 16:0 18:0 18:1 18:2 18:3 2692 RTW218 YLDGAT1 — 9.8 — — 13.16.4 31.7 38.6 10.2 2689 pKR1684 YLDGAT1 GmSut4-1 10.3 0.5 5.1% 13.7 6.833.8 35.5 10.2 2690 pKR1685 YLDGAT1 GmSut2-1 11.9 2.1 21.4% 13.1 6.1 322 39.2 9.5 2691 pKR1686 YLDGAT1 GmSut4-2 12.1 2.3 23.9% 12.9 6.7 32.538.6 9.3

In summary, co-expression of SUT2 and SUT4-type transporters, GmSut4-1,GmSut2-1 and GmSut4-2 showed significant additive effect by furtherincreasing oil contents compared to transgenic embryos with only theYLDGAT1 gene.

Example 15 Co-expression of Sucrose Transporters and Yarrowia lipolyticaDGAT2 in Soy

The present example describes cloning soy Sut4 and Sut2 genes, as wellas the Arabidospis SUT4His gene, into soy expression vectors andco-expressing with the modified Yarrowia lipolytica DGAT2 gene(YLDGAT2mod) (SEQ ID NO: 185) in soy somatic embryos. In all cases, eachSut gene is under control of the soy annexin promoter and YLDGAT2 isunder control of the soy glycinin Gy1 promoter.

Construction of pKR1691 (YLDGAT2mod 35Hvg)

Plasmid pKR407 (described in PCT Int. Appl. WO 2008/124048 published onOct. 16, 2008) was digested with BamHI/HindIII and the fragmentcontaining the Gy1 promoter/NotI/LegA2 terminator cassette waseffectively cloned into the BamHI/HindIII fragment of pKR278 (describedin PCT Int. Appl. WO 2008/147935 published on Dec. 4, 2008) to producepKR1468 (SEQ ID NO: 162).

The NotI fragment of pKR1254 Y326F (described in PCT Int. Appl. WO2008/147935 published on Dec. 4, 2008), containing a modified Yarrowialipolytica DGAT2 (YLDGAT2mod), was cloned into the Notl fragment ofpKR1468 (SEQ ID NO: 162), containing the vector backbone with Gy1promoter, to produce pKR1691 (SEQ ID NO: 163).

Construction of pKR1698 (ATSUT4HIS6 YLDGAT2mod 35Hyg)

The PstI fragment of RTW221 (SEQ ID NO: 134), containing ATSUT4HIS6, wascloned into the SbfI site of pKR1691 (SEQ ID NO: 163) to produce pKR1698(SEQ ID NO: 164).

Construction of pKR1699 (GmSut4-1 YLDGAT2mod 35Hyg)

The PstI fragment of pKR1680 (SEQ ID NO: 152), containing GmSut4-1, wascloned into the SbfI site of pKR1691 (SEQ ID NO: 163) to produce pKR1699(SEQ ID NO: 165).

Construction of pKR1700 (GmSut2-1 YLDGAT2mod 35Hyg)

The PstI fragment of pKR1681 (SEQ ID NO: 155), containing GmSut2-1, wascloned into the SbfI site of pKR1691 (SEQ ID NO: 163) to produce pKR1700(SEQ ID NO: 166).

Construction of pKR1701 (GmSut4-2 YLDGAT2mod 35Hyg)

The SbfI fragment of pKR1682 (SEQ ID NO: 160), containing GmSut4-2, wascloned into the SbfI site of pKR1691 (SEQ ID NO: 163) to produce pKR1701(SEQ ID NO: 167).

Transgenic soybean lines (cv. Jack) transformed with pKR1691 (SEQ ID NO:163), pKR1698 (SEQ ID NO: 164), pKR1699 (SEQ ID NO: 165), pKR1700 (SEQID NO: 166) or pKR1701 (SEQ ID NO: 167), were generated by particle gunbombardment as described in Example 7. Events were selected, somaticembryos were matured in SHaM media and embryos were analyzed for oilcontent and fatty acid profile exactly as described in Example 14.

Results showing oil content and fatty acid profile for approximately 30transgenic soybean lines (events) from each experiment transformed withpKR1698 (SEQ ID NO: 164), pKR1699 (SEQ ID NO: 165), pKR1700 (SEQ ID NO:166) or pKR1701 (SEQ ID NO: 167) are shown in Tables 13, 14, 15, 16 or17, respectively. Average oil content and fatty acid profile for allevents in an experiment is shown in each table as Avg. Average oilcontent and fatty acid profile for 5 events having highest oil contentin an experiment is shown in each Table as Avg-Top5. In each table,events are sorted based on decreasing oil content.

TABLE 13 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1691 (YLDGAT2mod only). MSE2702 (pKR1691) -YLDGAT2mod Event Oil 16:0 18:0 18:1 18:2 18:3 2702-10 10.1 12.7 5.8 32.438.8 10.3 2702-29 8.5 14.0 5.7 25.1 42.0 13.2 2702-5 8.1 13.9 5.6 25.940.6 14.0 2702-27 7.1 14.1 5.3 27.1 39.5 13.9 2702-16 6.9 14.6 5.5 27.338.5 14.1 2702-23 6.9 15.6 4.9 18.6 44.7 16.1 2702-7 6.7 13.7 7.3 31.135.3 12.6 2702-2 6.7 15.4 4.8 16.6 45.8 17.4 2702-9 5.7 17.2 5.7 19.241.8 16.1 2702-21 5.5 15.4 5.1 20.9 41.4 17.3 2702-4 5.4 15.3 6.3 25.037.7 15.7 2702-28 5.3 17.9 5.7 19.5 39.7 17.2 2702-18 5.2 16.3 5.1 16.742.5 19.5 2702-14 4.9 15.0 5.1 16.8 43.5 19.5 2702-13 4.9 16.6 4.5 15.843.3 19.8 2702-24 4.8 16.1 6.3 20.1 40.9 16.5 2702-20 4.7 15.7 5.7 21.039.9 17.7 2702-15 4.3 17.0 4.9 15.1 42.4 20.7 2702-6 4.3 16.4 5.6 17.441.9 18.6 2702-12 4.1 15.5 6.5 22.7 37.3 18.1 2702-26 4.1 16.1 4.7 15.342.3 21.6 2702-1 3.9 16.2 6.1 19.7 39.5 18.4 2702-22 3.8 17.1 5.7 17.940.5 18.8 2702-3 3.6 16.3 4.9 14.5 42.3 22.0 2702-8 3.6 17.7 5.0 15.240.8 21.2 2702-25 3.4 17.6 5.3 16.4 40.0 20.6 2702-17 3.3 17.3 5.3 17.937.8 21.8 2702-30 3.1 18.1 5.4 17.0 38.5 21.0 2702-19 3.0 17.8 5.7 17.039.4 20.0 2702-11 3.0 18.1 5.4 15.3 40.7 20.5 Avg. 5.2 16.0 5.5 20.040.7 17.8 Avg.-Top5 8.1 13.9 5.6 27.6 39.9 13.1

TABLE 14 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1698 (YLDGAT2mod & ATSUT4HIS6). MSE2703 (pKR1698) -YLDGAT2mod & AtSUT4HIS6 Event Oil 16:0 18:0 18:1 18:2 18:3 2703-2 12.512.4 5.2 34.3 39.2 8.9 2703-13 11.5 13.2 4.5 29.3 41.3 11.8 2703-22 9.913.3 5.6 30.0 40.2 10.9 2703-3 8.9 13.4 6.4 30.0 39.3 11.0 2703-7 7.913.5 5.9 30.8 37.5 12.3 2703-27 7.8 15.0 5.8 29.0 37.1 13.1 2703-26 7.213.8 6.8 32.7 35.0 11.6 2703-8 7.0 14.5 5.0 25.8 40.7 14.0 2703-14 6.214.4 6.0 28.3 37.4 14.0 2703-18 5.8 14.6 6.0 29.0 37.2 13.1 2703-15 5.817.7 4.9 18.2 42.9 16.2 2703-29 5.7 16.5 5.9 23.4 39.6 14.7 2703-6 5.216.0 4.8 18.4 42.0 18.9 2703-23 4.9 16.6 5.7 21.0 38.8 17.8 2703-9 4.917.6 4.5 17.1 42.7 18.1 2703-10 4.9 15.5 5.5 27.2 35.9 15.9 2703-16 4.417.0 5.3 20.2 40.3 17.2 2703-17 4.3 16.7 6.1 22.6 38.4 16.2 2703-5 4.116.8 6.0 19.7 42.6 15.0 2703-21 4.0 18.8 4.1 17.3 38.0 21.9 2703-24 4.015.7 4.4 20.0 40.3 19.6 2703-1 3.7 14.9 5.0 17.6 39.9 22.5 2703-11 3.717.4 6.1 22.6 35.8 18.1 2703-28 3.5 17.5 5.3 17.8 39.5 19.9 2703-19 3.513.3 5.4 17.3 43.3 20.7 2703-30 3.3 16.8 5.6 18.6 40.3 18.8 2703-12 3.316.6 5.3 23.0 34.0 21.0 2703-25 3.2 16.7 5.2 18.6 38.4 21.1 2703-4 3.116.5 5.7 20.3 37.8 19.7 2703-20 2.6 17.2 4.8 15.0 42.1 20.9 Avg. 5.615.7 5.4 23.2 39.2 16.5 Avg.-Top5 10.1 13.2 5.5 30.9 39.5 11.0

TABLE 15 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1699 (YLDGAT2mod & GmSut4-1). MSE2704 (pKR1699) -YLDGAT2mod & GmSut4-1 Event Oil 16:0 18:0 18:1 18:2 18:3 2704-10 16.310.8 4.8 38.0 38.6 7.8 2704-19 12.6 12.4 5.2 33.5 40.4 8.5 2704-21 10.713.7 4.5 27.5 42.3 11.9 2704-30 9.1 15.5 4.8 21.8 44.7 13.3 2704-6 8.914.0 6.4 35.3 33.6 10.7 2704-12 8.0 13.7 6.5 30.6 38.1 11.2 2704-20 7.714.1 6.0 30.5 36.6 12.9 2704-26 7.7 13.9 5.9 30.3 37.5 12.4 2704-9 7.315.1 6.0 24.8 39.6 14.6 2704-22 6.6 15.2 6.3 27.8 38.1 12.7 2704-23 6.017.0 4.9 19.6 42.3 16.2 2704-28 5.8 16.7 4.5 15.3 45.4 18.1 2704-2 5.716.5 5.4 16.9 43.0 18.2 2704-15 5.7 17.2 5.2 17.6 43.1 16.9 2704-18 5.316.0 5.3 23.2 39.0 16.4 2704-29 5.2 16.9 6.0 27.3 35.8 14.1 2704-7 5.216.3 5.4 19.7 42.3 16.5 2704-17 5.1 17.2 4.8 15.6 43.8 18.5 2704-11 5.015.2 5.6 18.1 42.6 18.5 2704-5 4.9 16.8 5.6 20.9 39.6 17.1 2704-8 4.816.6 6.3 24.4 34.9 17.8 2704-1 4.8 16.4 5.5 21.1 39.6 17.3 2704-16 4.717.4 4.9 16.8 42.2 18.7 2704-25 4.7 19.0 5.1 18.4 38.7 18.9 2704-27 4.618.7 5.8 19.8 37.9 17.8 2704-4 4.6 15.1 6.4 25.5 38.0 15.0 2704-24 4.516.8 5.4 19.1 40.7 18.0 2704-13 4.5 18.1 4.9 15.2 42.8 19.0 2704-3 3.716.7 6.0 22.5 37.0 17.8 2704-14 3.0 17.8 5.8 18.1 38.0 20.3 Avg. 6.415.9 5.5 23.2 39.9 15.6 Avg.-Top5 11.5 13.3 5.1 31.2 39.9 10.4

TABLE 16 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1700 (YLDGAT2mod & GmSut2-1). MSE2705 (pKR1700) -YLDGAT2mod & GmSut2-1 Event Oil 16:0 18:0 18:1 18:2 18:3 2705-9 10.513.6 6.2 32.3 38.2 9.6 2705-16 9.5 15.6 4.4 19.3 48.5 12.1 2705-1 9.515.5 5.8 21.2 45.9 11.6 2705-3 9.3 14.4 7.1 31.5 37.3 9.8 2705-8 9.213.7 6.1 29.8 40.0 10.4 2705-12 8.6 12.9 5.7 30.9 39.6 10.9 2705-27 8.413.6 5.3 29.9 39.2 12.0 2705-7 7.8 15.3 6.0 20.9 44.5 13.3 2705-17 7.416.6 7.5 23.6 40.0 12.3 2705-6 7.3 15.6 6.9 22.9 42.9 11.7 2705-5 7.116.9 5.1 19.7 44.0 14.2 2705-23 6.6 16.8 5.3 19.0 43.4 15.6 2705-13 6.415.4 6.1 21.3 41.7 15.6 2705-30 6.2 13.9 6.3 27.3 38.5 14.1 2705-29 6.216.1 7.2 22.8 38.9 15.0 2705-10 5.9 14.1 6.3 29.5 37.3 12.9 2705-28 5.817.1 5.3 19.9 42.5 15.2 2705-11 5.6 15.9 7.6 22.7 39.8 14.0 2705-15 5.616.6 5.9 19.7 42.2 15.7 2705-19 5.3 16.6 5.7 22.4 40.4 15.0 2705-2 5.215.1 4.8 15.9 46.5 17.7 2705-21 4.9 16.5 6.6 24.4 38.5 14.0 2705-22 4.916.3 5.1 18.6 41.5 18.4 2705-14 4.9 16.0 7.2 20.5 40.0 16.2 2705-4 4.818.8 6.1 21.0 38.7 15.5 2705-31 4.7 17.3 6.7 18.7 40.3 17.0 2705-24 4.517.2 4.9 17.0 42.1 18.9 2705-20 4.1 16.8 6.5 20.3 38.5 17.9 2705-25 4.114.8 4.3 16.1 42.8 22.0 2705-18 3.7 17.5 4.0 16.5 42.9 19.1 2705-26 3.717.8 6.0 18.4 39.3 18.4 Avg. 6.4 15.8 5.9 22.4 41.2 14.7 Avg.-Top5 9.614.6 6.0 26.8 42.0 10.7

TABLE 17 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1701 (YLDGAT2mod & GmSut4-2). MSE2706 (pKR1701) -YLDGAT2mod & GmSut4-2 Event Oil 16:0 18:0 18:1 18:2 18:3 2706-13 10.911.2 8.3 38.7 34.6 7.2 2706-16 8.2 14.0 7.0 33.3 36.3 9.4 2706-6 7.914.3 6.7 28.9 39.0 11.1 2706-2 7.7 16.2 4.0 19.7 45.4 14.8 2706-3 7.314.9 6.3 28.9 37.9 12.1 2706-1 6.5 12.7 5.7 34.3 36.1 11.2 2706-29 6.317.4 5.6 17.9 40.8 18.3 2706-24 5.9 15.1 6.8 27.8 36.7 13.6 2706-19 5.815.4 6.6 27.5 37.1 13.4 2706-10 5.8 17.1 5.9 26.2 35.5 15.2 2706-27 5.816.1 5.0 21.3 41.5 16.1 2706-26 5.7 15.8 6.2 26.9 37.3 14.0 2706-4 5.715.8 5.1 18.8 38.8 21.5 2706-30 5.6 16.2 7.0 26.4 36.6 13.8 2706-20 5.517.7 6.3 21.0 37.7 17.3 2706-28 5.3 15.3 5.4 26.0 39.9 13.4 2706-18 5.115.2 5.6 25.3 39.3 14.6 2706-8 4.9 16.9 6.0 24.2 36.4 16.5 2706-22 4.619.3 4.6 17.5 39.7 19.0 2706-14 4.6 17.3 6.5 23.3 37.7 15.3 2706-25 4.518.1 4.9 17.7 41.3 18.0 2706-12 4.5 18.5 5.0 15.9 39.6 21.1 2706-9 4.216.5 5.3 24.4 36.8 17.0 2706-17 4.0 17.0 6.5 22.2 36.2 18.1 2706-7 3.717.6 6.4 22.1 37.3 16.6 2706-15 3.5 17.8 6.2 21.2 36.0 18.7 2706-21 3.317.8 6.0 18.1 39.1 18.9 2706-11 3.3 17.5 4.9 18.1 38.5 20.9 2706-23 2.817.7 4.9 15.5 40.2 21.7 2706-5 2.5 16.3 5.7 14.4 35.9 27.7 Avg. 5.4 16.35.9 23.4 38.2 16.2 Avg.-Top5 8.4 14.1 6.4 29.9 38.6 10.9

A summary of the average oil content and fatty acid profile for the 5events having highest oil content for each experiment is shown in Table18. Also shown is the change in oil content compared to the YLDGAT1 onlyexperiment (dOil) as well as the percent increase in oil compared to theYLDGAT1 only experiment (% dOil).

TABLE 18 Average Oil Content and Fatty Acid Profiles for 5 Events havinghighest oil content in each experiment. Experiment Vector Gene 1 Gene 2Oil dOil % dOil 16:0 18:0 18:1 18:2 18:3 MSE2702 pKR1691 YLDGAT2mod —8.1 — — 13.9 5.6 27.6 39.9 13.1 MSE2703 pKR1698 YLDGAT2mod AtSUT4HIS610.1 2.0 24.7% 13.2 5.5 30.9 39.5 11 MSE2704 pKR1699 YLDGAT2mod GmSut4-111.5 3.4 42.0% 13.3 5.1 31.2 39.9 10.4 MSE2705 pKR1700 YLDGAT2modGmSut2-1 9.6 1.5 18.5% 14.6 6.0 26.8 42.0 10.7 MSE2706 pKR1701YLDGAT2mod GmSut4-2 8.4 0.3 3.7% 14.1 6.4 29.9 38.6 10.9

In summary, co-expression of SUT2 and SUT4-type transporters,ATSUT4HIS6, GmSut4-1 and GmSut2-1 showed significant additive effect byfurther increasing oil contents compared to transgenic embryos with onlythe YLDGAT2mod gene.

Example 16 Co-Expression of Sucrose Transporters and Yarrowia lipolyticaDGAT2 in Soy

The present example describes cloning a soy Sut4 gene (GmSut4-1) into asoy expression vector and co-expressing with the modified Yarrowialipolytica DGAT2 gene (YLDGAT2mod) in soy somatic embryos. GmSut4-1 isunder control of the soy beta-conglycinin promoter and YLDGAT2mod isunder control of the soy glycinin Gy1 promoter.

Construction of pKR1602 (YLDGAT2mod ALS)

Plasmid QC477 (described in US Publication No. US 2010-0162436 publishedon Jun. 24, 2010), which contains the soybean acetolactate synthase(als) gene coding region encoding a mutant ALS enzyme insensitive tosulfonylurea herbicides, was digested with NotI, the fragment ends werefilled to blunt and the fragment was religated to produce pKR1363 (SEQNO: 168). In this way, the NotI site was effectively removed.

The NotI fragment of pKR1316 (described in PCT Int. Appl. WO 2009/143401published on Nov. 26, 2009), containing a codon optimized Yarrowia DGAT1(YLDGAT1cod2), was cloned into the NotI fragment of pKR1104 (describedin PCT Int. Appl. WO 2008/124048 published on Oct. 16, 2008), containingthe vector backbone with soy beta-conglycinin promoter, to producepKR1331 (SEQ ID NO: 169).

Plasmid pKR1331 (SEQ ID NO: 169) was digested with BamHI/HindIII,fragment ends were completely filled to blunt and the fragmentcontaining YLDGAT1cod2 was then cloned into the PmeI site of pKR1363(SEQ ID NO: 168) to produce pKR1365 (SEQ ID NO: 170).

The NotI fragment of pKR1254 Y326F (described in PCT Int. Appl. WO2008/147935 published on Dec. 4, 2008), containing a modified YarrowiaDGAT2 (YLDGAT2mod), was cloned into the NotI fragment of pKR1365 (SEQ IDNO: 170), containing the vector backbone with soy beta-conglycininpromoter, to produce pKR1374 (SEQ ID NO: 171).

The XbaI/NheI fragment of pKR263 (described in PCT Int. Appl. WO2004/071467 published on Aug. 26, 2004), containing the Gy1 promoter/legterminator cassette, was cloned into the XbaI site of pNEB193 (NewEngland Biolabs, Ipswich, Mass.) to produce pKR1598 (SEQ ID NO: 172).

The NotI fragment of pKR1374 (SEQ ID NO: 171), containing YLDGAT2mod,was cloned into the NotI site of pKR1598 (SEQ ID NO: 172) to producepKR1600 (SEQ ID NO: 173).

The SbfI/BsiWI fragment of pKR1600 (SEQ ID NO: 173), containingYLDGAT2mod, was cloned into the SbfI/BsiWI fragment of pKR1374 (SEQ IDNC): 171), containing the vector backbone with ALS selection marker, toproduce pKR1602 (SEQ ID NO: 174).

Construction of pKR1661 (GmSut4-1 YLDGAT2mod ALS).

The NotI fragment of Glyma02g38300 in pGEM-T Easy (SEQ ID NO: 151),containing GmSut4-1, was cloned into the NotI site of pKR1365 (SEQ IDNO: 170) to produce pKR1658 (SEQ ID NO: 175).

The BsiWI fragment of pKR1658 (SEQ ID NO: 175), containing GmSut4-1, wascloned into the BsiWI site of pKR1602 (SEQ ID NO: 174) to producepKR1661 (SEQ ID NO: 176).

Transgenic soybean lines transformed with pKR1602 (SEQ ID NO: 174) orpKR1661 (SEQ ID NO: 176), were generated by particle gun bombardment asdescribed in Example 7 with the following modifications. Soy embryogenicsuspension cultures (cv. 93B86) were grown at a light intensity of80-100 μE/m²/s and were subcultured every 7-14 days by inoculating up to½ dime sized quantities of tissue into 50 mL of fresh liquid SB196.During tissue bombardment, only 100 mg of two-week old suspensionculture is transformed (vs 150-250 mg described in Example 7). Totaltime in selection on SU is for 13 weeks vs 8-12 as described in Example7.

Events were analyzed at the somatic embryo stage for oil content andfatty acid profile exactly as described in Example 7. Results showingoil content and fatty acid profile for transgenic soybean lines (events)from each experiment transformed with pKR1602 (SEQ ID NO: 174) orpKR1661 (SEQ ID NO: 176) are shown in Tables 19 or 20, respectively.Average oil content and fatty acid profile for all events in anexperiment is shown in each table as Avg. Average oil content and fattyacid profile for 5 events having highest oil content in an experiment isshown in each Table as Avg-Top5. In each table, events are sorted basedon decreasing oil content.

TABLE 19 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1602 (YLDGAT2mod). Oil60 (pKR1602) - YLDGAT2modEvent Oil 16:0 18:0 18:1 18:2 18:3 AFS 7220-11-2 8.7 15.3 5.2 15.1 51.712.7 AFS 7501-12-1 8.0 12.7 4.3 29.4 40.7 13.0 AFS 7220-9-1 7.7 13.7 7.816.9 47.8 13.7 AFS 7501-10-1 7.3 12.1 5.0 30.7 39.6 12.6 AFS 7501-7-17.0 15.3 4.0 20.8 46.4 13.5 AFS 7220-1-1 6.6 13.3 7.2 30.8 39.5 9.3 AFS7501-1-1 6.3 14.4 4.4 27.5 40.3 13.4 AFS 7220-4-1 5.4 14.2 7.9 17.9 46.513.6 AFS 7220-10-1 4.5 13.1 8.0 26.2 42.0 10.7 AFS 7501-12-2 4.2 14.54.4 24.9 40.4 15.7 AFS 7501-6-1 4.2 15.8 4.3 22.4 40.9 16.6 AFS7220-10-2 3.8 17.0 6.5 19.5 43.8 13.1 AFS 7501-12-3 3.6 15.8 5.0 21.439.7 18.0 AFS 7220-11-1 3.6 13.5 6.6 19.0 47.6 13.4 Avg. 5.8 14.3 5.823.0 43.3 13.5 Avg.-Top5 7.7 13.8 5.3 22.6 45.2 13.1

TABLE 20 Oil Content and Fatty Acid Profile for Soy Somatic EmbryosTransformed with pKR1661 (YLDGAT2mod & GmSut4-1). Oil79 (pKR1661) -YLDGAT2mod & GmSut4-1 Event Oil 16:0 18:0 18:1 18:2 18:3 AFS 7162-10-1611.4 14.3 5.5 29.4 39.0 11.8 AFS 7162-10-15 9.8 11.3 5.4 37.5 36.8 9.0AFS 7162-10-4 9.0 10.2 4.3 33.0 41.8 10.7 AFS 7162-10-1 8.8 10.8 5.334.8 39.1 9.9 AFS 7162-10-7 8.5 14.9 4.5 23.6 45.7 11.3 AFS 7162-5-2 8.211.8 6.0 30.0 40.1 12.0 AFS 7162-10-5 8.1 10.3 5.0 40.2 35.4 9.1 AFS7162-10-17 8.0 12.6 6.5 35.7 35.0 10.2 AFS 7162-10-11 7.3 12.1 5.1 30.439.1 13.2 AFS 7162-10-14 7.3 15.3 5.7 23.2 37.9 17.9 AFS 7162-10-2 7.310.6 6.2 41.6 32.3 9.4 AFS 7162-10-9 7.0 11.1 5.0 35.5 36.8 11.5 AFS7162-10-12 6.6 12.4 6.0 41.9 30.3 9.4 AFS 7162-10-10 6.4 11.8 5.2 33.438.5 11.2 AFS 7162-10-3 5.7 12.5 4.3 21.7 44.4 17.0 AFS 7162-10-6 5.517.4 4.3 17.0 45.0 16.3 AFS 7162-5-1 5.5 12.6 5.7 25.5 41.7 14.6 AFS7162-10-8 5.4 14.2 4.3 25.8 40.4 15.3 AFS 7162-10-13 5.4 13.2 4.4 21.844.8 15.9 AFS 7162-5-3 5.3 13.2 7.5 33.4 31.8 14.0 Avg. 7.3 12.6 5.330.8 38.8 12.5 Avg.-Top5 9.5 12.3 5.0 31.7 40.5 10.6

A summary of the average oil content and fatty acid profile for the 5events having highest oil content for each experiment is shown in Table21. Also shown is the change in oil content compared to the YLDGAT1 onlyexperiment (dOil) as well as the percent increase in oil compared to theYLDGAT1 only experiment (% dOil).

TABLE 21 Average Oil Content and Fatty Acid Profiles for 5 Events havinghighest oil content in each experiment. Average Oil Content and FattyAcid Profile for Top5 Events per Experiment Experiment Vector Gene 1Gene 2 Oil dOil % dOil 16:0 18:0 18:1 18:2 18:3 Oil60 pKR1602 YLDGAT2mod— 7.7 — — 13.8 5.3 22.6 45.2 13.1 Oil79 pKR1661 YLDGAT2mod GmSut4-1 9.51.8 23.3% 12.3 5.0 31.7 40.5 10.6

In summary, co-expression of GmSut4-1 showed significant additive effectby further increasing oil contents compared to transgenic embryos withonly the YLDGAT2mod gene.

Example 17 Co-Expression of Yarrowia lipolytica DGAT Genes and LowAffinity, High Capacit Sucrose Transporter Genes in Soybean Seed

T1 seed from event AFS 5925.1.9.2 (3 transgenic HiOil/Increased Oleicsegregants; 1 null segregant) and AFS 5925.2.7.1 (9 transgenicHiOil/Increased Oleic segregants; 2 null segregants), which weredescribed in Example 8 (Table 7), were planted and plants grown asdescribed supra. T2 seed were harvested and 16 seeds from each seed wereanalyzed for oil content and fatty acid profile as described supra.Based on the oil content and fatty acid profiles for T2 seed from eachplant, 1 plant from event AFS 5925.1.9.2 and 1 plant from event AFS5925.2.7.1 were identified to be homozygous for the transgene as all 16seed analyzed displayed the HiOil/Increased Oleic phenotype.

The oil content and fatty acid profiles for seed from homozygous plants(Homoz) as well as the null segregants (Null) for each event are shownin Table 22. In Table 22, “Avg.” indicates the average oil content orfatty acid profile for all T2 seed from that plant. In Table 22, NullAvg. indicates the average oil content and fatty acid profile for T2seed from all Null plants from AFS 5925.2.7.1. Data are presented asdecreasing oleic acid content in each seed for each plant. Oil contentfrom 2 wild-type soybean seed (cv. Jack) is also shown.

TABLE 22 Oil Content and Fatty Acid Profile of T2 Soybean Seed Generatedwith PHP 36710 Segregant Event Plant Seed % oil %16:0 %18:0 %18:1 %18:2%18:3 Type AFS 5925.1.9.2 9GR22-74 9GR22-74-1 20.4 12.6 3.7 14.8 57.411.5 Null AFS 5925.1.9.2 9GR22-74 9GR22-74-16 21.2 13.4 3.6 14.3 58.210.5 Null AFS 5925.1.9.2 9GR22-74 9GR22-74-12 20.9 12.9 4.2 13.7 59.69.6 Null AFS 5925.1.9.2 9GR22-74 9GR22-74-7 21.8 12.1 4.1 13.5 60.9 9.5Null AFS 5925.1.9.2 9GR22-74 9GR22-74-13 21.8 12.5 3.8 13.0 60.1 10.7Null AFS 5925.1.9.2 9GR22-74 9GR22-74-10 18.5 13.5 3.5 12.9 58.1 12.0Null AFS 5925.1.9.2 9GR22-74 9GR22-74-8 20.7 13.4 3.6 12.6 59.6 10.8Null AFS 5925.1.9.2 9GR22-74 9GR22-74-11 20.4 12.9 3.7 12.3 60.1 11.1Null AFS 5925.1.9.2 9GR22-74 9GR22-74-4 20.1 13.0 4.0 12.1 58.0 13.0Null AFS 5925.1.9.2 9GR22-74 9GR22-74-2 20.2 13.2 3.7 10.9 58.2 14.0Null AFS 5925.1.9.2 9GR22-74 9GR22-74-9 21.2 12.6 3.7 10.8 60.0 12.9Null AFS 5925.1.9.2 9GR22-74 9GR22-74-5 20.6 12.7 3.6 10.6 60.6 12.6Null AFS 5925.1.9.2 9GR22-74 9GR22-74-15 20.7 12.5 3.8 10.2 59.0 14.5Null AFS 5925.1.9.2 9GR22-74 9GR22-74-14 20.5 13.4 3.9 10.1 55.5 17.1Null AFS 5925.1.9.2 9GR22-74 9GR22-74-3 15.8 13.7 3.6 10.0 59.6 13.1Null AFS 5925.1.9.2 9GR22-74 9GR22-74-6 12.7 15.4 3.8 8.8 52.0 20.0 Null9GR22-74 19.8 13.1 3.8 11.9 58.5 12.7 Avg. AFS 5925.1.9.2 9GR22-939GR22-93-15 27.0 10.5 5.3 37.2 42.2 4.8 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-4 18.9 9.0 4.9 36.4 43.4 6.3 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-14 27.7 10.5 5.4 34.2 44.3 5.6 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-16 24.9 11.1 5.1 33.2 44.9 5.7 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-3 26.5 10.8 4.8 32.7 46.2 5.5 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-5 27.8 10.3 5.2 32.7 45.8 6.1 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-12 26.1 11.4 4.8 31.1 46.9 5.8 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-10 26.0 11.6 5.7 30.9 46.1 5.7 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-7 26.0 12.3 5.9 30.6 44.4 6.7 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-6 24.4 11.5 4.8 29.7 48.0 6.0 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-9 25.8 11.3 6.0 29.5 47.0 6.3 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-2 27.0 11.3 5.0 29.5 47.8 6.5 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-8 24.8 11.0 4.4 29.3 49.6 5.7 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-13 27.9 11.2 4.8 29.3 48.0 6.7 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-1 24.5 11.8 4.8 28.6 49.3 5.4 Homoz AFS 5925.1.9.2 9GR22-939GR22-93-11 25.6 12.3 5.3 27.6 48.4 6.4 Homoz AFS 5925.1.9.2 9GR22-9325.7 11.1 5.1 31.4 46.4 5.9 Avg. AFS 5925.2.7.1 9GR22-114 9GR22-114-320.8 11.1 3.5 13.8 63.4 8.2 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1220.1 12.4 4.0 12.5 60.1 10.9 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-620.6 11.4 3.5 11.9 64.1 9.1 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1321.0 12.1 3.7 11.8 62.8 9.6 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1019.6 13.0 3.5 11.2 60.2 12.1 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-220.6 11.5 3.5 11.0 61.9 12.0 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1417.9 12.8 3.4 10.7 59.3 13.7 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1123.2 10.6 3.4 10.6 63.2 12.2 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-718.9 11.5 3.5 10.0 64.4 10.7 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-119.4 10.7 3.4 9.9 65.2 10.7 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-820.3 13.0 3.3 9.6 60.8 13.3 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1521.7 11.1 3.3 9.2 63.0 13.3 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-922.4 10.6 3.2 7.9 65.4 12.8 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-519.6 11.9 3.2 7.1 58.0 19.8 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-419.1 11.8 3.8 6.9 62.1 15.3 Null AFS 5925.2.7.1 9GR22-114 9GR22-114-1619.7 10.4 3.4 6.1 61.2 19.0 Null AFS 5925.2.7.1 9GR22-114 20.3 11.6 3.510.0 62.2 12.7 Avg. AFS 5925.2.7.1 9GR22-115 9GR22-115-15 21.4 12.1 3.513.8 61.5 9.2 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-7 21.7 11.5 3.612.2 63.1 9.6 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-14 21.2 11.6 3.311.9 63.9 9.3 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-16 19.9 11.0 3.811.7 63.3 10.2 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-3 21.5 11.5 3.311.6 63.8 9.7 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-13 21.3 10.7 4.211.4 63.0 10.7 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-12 22.0 11.9 3.611.1 62.2 11.3 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-5 22.1 11.5 4.511.0 61.7 11.3 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-6 21.0 11.3 3.810.7 63.6 10.6 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-1 20.3 11.1 3.910.5 63.7 10.9 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-2 21.5 12.5 3.79.6 62.3 11.9 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-8 21.0 12.8 4.09.5 61.9 11.8 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-9 21.0 11.4 3.89.0 64.3 11.4 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-10 21.3 12.5 3.38.8 63.0 12.4 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-11 20.0 12.2 3.98.8 58.6 16.5 Null AFS 5925.2.7.1 9GR22-115 9GR22-115-4 19.7 11.1 3.37.9 63.3 14.3 Null AFS 5925.2.7.1 9GR22-115 21.1 11.7 3.7 10.6 62.7 11.3Avg. Null Avg. 20.7 11.6 3.6 10.3 62.5 12.0 AFS 5925.2.7.1 9GR22-1339GR22-133-6 24.7 11.6 6.1 29.8 47.9 4.7 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-11 25.0 11.4 6.9 28.3 49.0 4.5 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-9 25.0 11.9 5.6 27.8 49.7 5.0 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-10 25.4 11.4 6.4 27.5 50.5 4.2 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-3 24.1 12.0 6.4 27.3 49.8 4.5 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-13 26.2 11.1 6.3 27.2 49.9 5.5 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-12 24.9 11.8 6.4 27.0 50.2 4.6 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-2 25.9 11.8 5.9 26.2 51.3 4.8 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-16 26.6 12.7 6.5 25.6 50.2 5.0 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-5 26.2 11.7 7.0 25.6 50.4 5.3 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-4 23.7 12.7 6.3 25.5 51.1 4.4 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-15 25.5 11.9 6.7 24.5 51.7 5.2 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-1 25.4 13.0 6.0 24.4 50.8 5.8 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-8 27.1 12.6 6.4 22.4 52.4 6.1 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-14 24.1 13.3 6.2 21.3 53.2 6.0 Homoz AFS 5925.2.7.1 9GR22-1339GR22-133-7 24.8 13.5 6.0 20.5 53.2 6.7 Homoz AFS 5925.2.7.1 9GR22-13325.3 12.2 6.3 25.7 50.7 5.1 Avg. Jack 9GR22-425 9GR22-425-12 20.9 12.84.3 12.4 60.1 10.5 N/A Jack 9GR22-425 9GR22-425-4 20.8 13.0 4.2 12.259.8 10.8 N/A 9GR22-425 24.8 12.4 6.1 23.2 52.3 6.0 Avg.

A summary of the average oil contents and fatty acid profiles fromtransgenic homozygous T2 seed compared to null T2 seed for events AFS5925.1.9.2 and AFS 5925.2.7.1, is shown in Table 23. Also shown in Table23 is the difference in average oil content and fatty acid between thetransgenic homozygous seed and the null seed for each event (delta). Theaverage % change for the transgenic homozygous seed compared to the nullis also shown (% delta).

TABLE 23 T2 Seed Event Type % oil %16:0 %18:0 %18:1 %18:2 %18:3 AFS5925.1.9.2 Homoz Avg. 25.7 11.1 5.1 31.4 46.4 5.9 AFS 5925.1.9.2 NullAvg. 19.8 13.1 3.8 11.9 58.5 12.7 delta 5.9 −2.0 1.4 19.5 −12.2 −6.7 %delta 29% −15% 37% 164% −21% −53% AFS 5925.2.7.1 Homoz Avg. 25.3 12.26.3 25.7 50.7 5.1 AFS 5925.2.7.1 Null Avg. 20.7 11.6 3.6 10.3 62.5 12.0delta 4.6 0.5 2.7 15.4 −11.8 −6.9 % delta 22%    4% 75% 149% −19% −57%

In summary applicants have demonstrated that co-expression of DGAT genesand SUT2 or SUT4 sucrose transporter genes provides an efficient methodto increase the total fatty acid content of seed.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A soybean plant seed with increased oilcontent comprising in its genome: (a) a first recombinant DNA constructcomprising a first polynucleotide operably linked to at least one firstregulatory element, wherein the first polynucleotide comprises adiacylglycerol acyltransferase (DGAT) coding region encoding apolypeptide having at least 95% sequence identity to SEQ ID NO:180,based on the Clustal W method of alignment with pairwise alignmentdefault parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5, wherein the DGAT polypeptide has DGAT activity; (b) a secondrecombinant DNA construct comprising a second polynucleotide operablylinked to at least one second regulatory element, wherein the secondpolynucleotide encodes a SUT4 sucrose transporter polypeptide, whereinthe SUT4 sucrose transporter polypeptide has at least 95% sequenceidentity to SEQ ID NO: 40, based on the Clustal W method of alignmentwith pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5; and (c) wherein the soybean plant seedexhibits increased oil content as a percentage of seed weight whencompared to a control soybean plant seed comprising the firstrecombinant DNA construct but not comprising the second recombinant DNAconstruct.
 2. The plant seed of claim 1, wherein the firstpolynucleotide encodes a polypeptide having the amino acid sequence ofSEQ ID NO:180, 184, 186, 188, or
 190. 3. The plant seed of claim 1,wherein the second polynucleotide a polypeptide having at least 95%sequence identity to SEQ ID NO: 40 based on the Clustal W method ofalignment with pairwise alignment default parameters of KTUPLE=1, GAPPENALY=3, WINDOW=5 and DIAGONALS SAVED=5.
 4. A soybean plant grown fromthe seed of claim
 1. 5. A method of increasing oil content in a soybeanplant seed comprising: (a) introducing into a regenerable soybean plantcell: (i) a first recombinant DNA construct comprising a firstpolynucleotide encoding a DGAT polypeptide having at least 95% sequenceidentity to SEQ ID NO:180, based on the Clustal W method of alignmentwith pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5, and (ii) a second recombinant DNAconstruct comprising a second polynucleotide encoding a SUT4 sucrosetransporter polypeptide, wherein the SUT4 sucrose transporterpolypeptide has at least 95% sequence identity to SEQ ID NO: 40, basedon the Clustal W method of alignment with pairwise default parameters ofKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome the firstrecombinant DNA construct and the second recombinant DNA construct; and,(c) obtaining a progeny plant derived from the transgenic plant of step(b), wherein the progeny plant comprises in its genome the firstrecombinant DNA construct and the second recombinant DNA construct,wherein the progeny plant exhibits increased oil content as a percentageof seed weight when compared to a control soybean plant comprising thefirst recombinant DNA construct but not comprising the secondrecombinant DNA construct.
 6. The method of claim 5, wherein the secondpolynucleotide encodes a polypeptide having the amino acid sequence ofSEQ ID NO:
 40. 7. The method of claim 5, wherein the recombinant DNAconstruct encoding the SUT4 polypeptide comprises an amino acid sequenceof at least 98% sequence identity to SEQ ID NO: 40 based on the ClustalW method of alignment with pairwise default parameters of KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
 8. The method of claim 5,wherein the first polynucleotide encodes a polypeptide having the aminoacid sequence of SEQ ID NO:180, 184, 186, 188 or
 190. 9. A method ofevaluating increased seed oil content in a soybean plant seedcomprising: evaluating seed from a progeny plant grown from a transgenicplant for increased oil content, the transgenic plant comprising in itsgenome: (i) a first recombinant DNA construct comprising a firstpolynucleotide encoding a DGAT polypeptide having at least 95% sequenceidentity to SEQ ID NO:180, based on the Clustal W method of alignmentwith pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5, wherein the DGAT polypeptide has DGATactivity; and (ii) a second recombinant DNA construct comprising asecond polynucleotide encoding a SUT4 sucrose transporter polypeptide,wherein the SUT4 sucrose transporter polypeptide has at least 95%sequence identity to SEQ ID NO: 40, based on the Clustal W method ofalignment with pairwise alignment default parameters of KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 wherein the progeny plantcomprises in its genome the first recombinant DNA construct and thesecond recombinant DNA construct; and wherein the seed from the progenyplant shows increased oil content compared to a control soybean plantnot comprising the first recombinant DNA construct and the secondrecombinant DNA construct.
 10. The method of claim 9, wherein the secondpolynucleotide encodes a polypeptide having an amino acid sequence ofSEQ ID NO:
 40. 11. The method of claim 9, wherein the firstpolynucleotide encodes a polypeptide having the amino acid sequence ofSEQ ID NO: 180, 184, 186, 188, or
 190. 12. The plant seed of claim 1,wherein the first regulatory element comprises a seed-specific promoter,and wherein the second regulatory element comprises a differentseed-specific promoter.
 13. The plant seed of claim 12, wherein thefirst seed-specific promoter, the second seed-specific promoter or acombination thereof is selected from the group consisting of the alphaprime subunit of beta conglycinin promoter, soybean sucrose synthasepromoter, kunitx trypsin inhibitor 3 promoter, annexin promoter, Gly1promoter, beta subunit of beta conglycinin promoter, P34/Gly Bd m 30 Kpromoter, albumin promoter, Leg A1 promoter and Leg A2 promoter.
 14. Theplant seed of claim 1, wherein the plant seed exhibits an overallincreased oil content of 1% or greater when compared to the controlplant.
 15. The plant seed of claim 1, wherein the plant seed exhibits anoverall increased oil content of 5% or greater when compared to thecontrol plant.
 16. The plant seed of claim 1, wherein the plant seedexhibits an overall increased oil content of 10% or greater whencompared to the control plant.
 17. The method of claim 5, wherein thefirst regulatory sequence comprises a seed-specific promoter, andwherein the second regulatory sequence comprises a differentseed-specific promoter.
 18. The method of claim 5, wherein the plantseed exhibits an overall increased oil content of 1% or greater whencompared to control plant.
 19. The method of claim 5, wherein the plantseed exhibits an overall increased oil content of 5% or greater whencompared to the control plant.
 20. The method of claim 5, wherein theplant seed exhibits an overall increased oil content of 10% or greaterwhen compared to the control plant.